Directed Evolution and In Vivo Panning of Virus Vectors

ABSTRACT

The present invention provides methods of achieving directed evolution of viruses by in vivo screening or “panning” to identify viruses comprising scrambled AAV capsids having characteristics of interest, e.g., tropism profile and/or neutralization profile (e.g., ability to evade neutralizing antibodies). The invention also provides scrambled AAV capsids and virus particles comprising the same.

RELATED APPLICATION INFORMATION

This application claims the benefit of U.S. Provisional Application No.61/049,160 filed Apr. 30, 2008, the disclosure of which is incorporatedby reference herein in its entirety.

STATEMENT OF FEDERAL SUPPORT

Aspects of this invention were supported with funding provided underGrant No. 2RO1AR 45967 awarded by the National Institutes of Health. TheUnited States Government has certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to methods for directed evolution and in vivopanning of adeno-associated virus vectors as well as optimized AAVcapsids and virus vectors comprising the same.

BACKGROUND OF THE INVENTION

The muscular dystrophies (MD) are a heterogeneous group of inheriteddisorders characterized by progressive weakness and degeneration ofskeletal muscles. The molecular basis of Duchenne muscular dystrophy(DMD) was first elucidated twenty years ago as a perturbation ofdystrophin (Koenig et al., (1987) Cell 50: 509-517). Dystrophinassociates with a number of proteins to form a large oligomeric complexnamed the dystrophin-glycoprotein complex (DGC), which bridges acrossthe sarcolemma and connects the extracellular matrix and the actincytoskeleton (Allamand and Campbell, (2000) Human Molecular Genetics9:2459-2467). Loss or abnormal function of the DGC components will leadto dystrophic muscles and various forms of MD. For instance, mutationsin sarcoglycan (SG) genes are responsible for autosomal recessivelimb-girdle muscular dystrophies (LGMD 2C-2F; reviewed by Lim andCampbell, (1998) Neurology 11:443-452), and deficiency in the laminin α2chain is responsible for about half of the cases of congenital musculardystrophy (CMD; Helbling-Leclerc et al., (1995) Nature Genetics11:216-218).

Adeno-associated virus (AAV) was first reported to efficiently transducemuscle over ten years ago (Xiao et al., (1996) J. Virology70:8098-8108). As another advantage, AAV vectors have a good safetyprofile. The recombinant AAV (rAAV) genome composed of a foreignexpression cassette and AAV inverted terminal repeat (ITR) sequencesexists in eukaryotic cells in an episomal form that is responsible forpersistent transgene expression (Schnepp et al., (2003) J. Virology77:3495-3504). No human disease has been associated with wild-type AAVinfection and low toxicity is observed in human subjects followingmuscle transduction by rAAV (Manno et al., (2003) Blood 101:2963-2972).

A series of new AAV serotypes have been identified from humans orprimates that display variable capsid sequences as compared with AAV2(Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854-11859; Gao etal., (2004) J. Virology 78:6381-6388). Of these, AAV1, 6, 8, and 9recombinant vectors have been reported to result in higher transgeneexpression level in muscle than rAAV2 vector (Wang et al., (2005) NatureBiotech. 23:321-328; Inagaki et al., (2006) Molecular Therapy 14:45-53).Widespread transduction of cardiac and skeletal muscle has been achievedin adult mouse by intravenous administration of rAAV6 vectorsupplemented with vascular endothelium growth factor (VEGF) (Gregorevicet al., (2004) Nature Med. 10:828-834). rAAV1 vectors have a similarcapsid sequence and were successfully applied in systemic gene deliveryto vital muscles of DMD and CMD mouse models and efficiently amelioratedthe dystrophic phenotype (Qiao et al., (2005) Proc. Nat. Acad. Sci. USA102:11999-12004; Denti et al., (2006) Proc. Nat. Acad. Sci. USA103:3758-3763). Whilst vector administration in these studies wasconducted with pharmacological interventions or in neonatal animals,rAAV8 vectors appear more efficient at crossing the blood vessel barrierand transducing heart and skeletal muscle of adult mice and hamsters(Wang et al., (2005) Nature Biotech. 23:321-328; Zhu et al., (2005)Circulation 112:2650-2659). AAV9 vectors also demonstrate efficienttropism to the myocardium and serve as another alternative for systemicgene delivery to heart (Inagaki et al., (2006) Molecular Therapy14:45-53; Pacak et al., (2006) Circulation Research 99:3-9).

In addition to the investigation of natural AAV serotypes, researchefforts have explored modification of the AAV capsid to produceoptimized vectors. Mutagenesis represented the initial approach togenetically modify the AAV2 capsid (Wu et al., (2000) J. Virology74:8635-8647; Lochrie et al., (2006) J. Virology 80:821-834). Insertionof peptides from phage display libraries into the AAV capsid proteinproved to be another strategy to modify the AAV capsid and retarget thevector to new cells or tissues. This method has been further developedto directly display synthesized peptides on the surface of the AAVcapsid.

Besides rational design, directed evolution has been used to introducemodifications into AAV vectors. One group reported that the AAV2 capsidgene was diversified by random mutagenesis and then subject to in vitrorecombination (Maheshri et al., (2006) Nature Biotech. 24:198-204). Themodified capsid genes were employed for the production of an AAVlibrary, which was screened in vitro for enhanced properties such asaltered affinities for heparin or evasion of antibody neutralization. Invitro screening methods, however, are inherently limited in theirability to identify optimized mutants that will have desired propertiesin vivo in the context of a complex biological system. For example, thevasculature is a major barrier for systemic AAV delivery via thecirculation to many tissues and cell types including skeletal muscle,diaphragm muscle, the heart and brain. It would be desirable for an AAVvector to not only be efficient in crossing the endothelial lining toreach the intended target cells such as cardiomyocytes in the heart,myofibers in skeletal muscle and neurons in the brain, but also berobust in infecting those intended cells after reaching them. An invitro panning system is simply unable to select for both of theseproperties. In addition, there are numerous examples in the literaturein which in vitro assessment of viral properties such as tropism was notpredictive of in vivo behavior.

SUMMARY OF THE INVENTION

The present invention provides methods of achieving directed evolutionof viruses by in vivo screening or “panning” to identify virusescomprising mosaic or “scrambled” AAV capsids having characteristics ofinterest, e.g., tropism profile and/or neutralization profile (e.g.,ability to evade neutralizing antibodies). The invention also providesscrambled AAV capsids and virus particles comprising the same.

Thus, as one aspect, the invention provides a nucleic acid (e.g., anisolated nucleic acid) encoding an AAV capsid, the nucleic acidcomprising an AAV capsid coding sequence selected from the groupconsisting of:

-   -   (a) the nucleotide sequence of FIG. 3E (M17) (SEQ ID NO:1);    -   (b) the nucleotide sequence of FIG. 3H (M22) (SEQ ID NO:3);    -   (c) the nucleotide sequence of FIG. 3K (M35) (SEQ ID NO:5);    -   (d) the nucleotide sequence of FIG. 3B (M41) (SEQ ID NO:7);    -   (e) the nucleotide sequence of FIG. 3N (M42) (SEQ ID NO:9);    -   (f) the nucleotide sequence of FIG. 3Q (M62) (SEQ ID NO:11);    -   (g) the nucleotide sequence of FIG. 3T (M67) (SEQ ID NO:13);    -   (h) the nucleotide sequence of FIG. 3W (M125) (SEQ ID NO:15);    -   (i) the nucleotide sequence of FIG. 3Z (M148) (SEQ ID NO:17);    -   (j) the nucleotide sequence of FIG. 3CC (M151) (SEQ ID NO:19);    -   (k) the nucleotide sequence of FIG. 3FF (H18) (SEQ ID NO:21);    -   (l) the nucleotide sequence of FIG. 3II (H34) (SEQ ID NO:23);    -   (m) the nucleotide sequence of FIG. 3LL (H39) (SEQ ID NO:25);    -   (n) the nucleotide sequence of FIG. 3OO (H40) (SEQ ID NO:27);    -   (o) the nucleotide sequence of FIG. 3RR (H43) (SEQ ID NO:29);    -   (p) the nucleotide sequence of FIG. 3UU (H50) (SEQ ID NO:31);    -   (q) the nucleotide sequence of FIG. 3XX (H53) (SEQ ID NO:33);    -   (r) the nucleotide sequence of FIG. 3AAA (H66) (SEQ ID NO:35);    -   (s) the nucleotide sequence of FIG. 3DDD (H109) (SEQ ID NO:37);    -   (t) the nucleotide sequence of FIG. 6A (HH1) (SEQ ID NO:39);    -   (u) the nucleotide sequence of FIG. 6C(HH15) (SEQ ID NO:41);    -   (vv) the nucleotide sequence of FIG. 6E (HH19) (SEQ ID NO:43);    -   (ww) the nucleotide sequence of FIG. 6G (HH27) (SEQ ID NO:45);    -   (xx) the nucleotide sequence of FIG. 6I (HH35) (SEQ ID NO:47);    -   (yy) the nucleotide sequence of FIG. 6K (HH41) (SEQ ID NO:49);    -   (zz) the nucleotide sequence of FIG. 6M (HH45) (SEQ ID NO:51);    -   (aaa) the nucleotide sequence of FIG. 6O (HH53) (SEQ ID NO:53);    -   (bbb) the nucleotide sequence of FIG. 6Q (HH67) (SEQ ID NO:55);    -   (ccc) the nucleotide sequence of FIG. 6S(HH68) (SEQ ID NO:57);    -   (ddd) the nucleotide sequence of FIG. 6U (HH75) (SEQ ID NO:59);    -   (eee) the nucleotide sequence of FIG. 6W (HH87) (SEQ ID NO:61);    -   (fff) the nucleotide sequence of FIG. 6Y (HH64) (SEQ ID NO:63);    -   (ggg) the nucleotide sequence of FIG. 6AA (MH4) (SEQ ID NO:65);    -   (hhh) the nucleotide sequence of FIG. 6CC (MH18) (SEQ ID NO:67);    -   (iii) the nucleotide sequence of FIG. 6EE (MH21) (SEQ ID NO:69);    -   (jjj) the nucleotide sequence of FIG. 6GG (MH31) (SEQ ID NO:71);    -   (kkk) the nucleotide sequence of FIG. 6II (MH39) (SEQ ID NO:73);    -   (lll) the nucleotide sequence of FIG. 6KK (MHY43) (SEQ ID        NO:75);    -   (mmm) the nucleotide sequence of FIG. 6MM (MH47) (SEQ ID NO:77);    -   (nnn) the nucleotide sequence of FIG. 6OO (MH58) (SEQ ID NO:79);    -   (ooo) the nucleotide sequence of FIG. 6QQ (MH63) (SEQ ID NO:81);    -   (ppp) the nucleotide sequence of FIG. 6SS (MH71) (SEQ ID NO:83);    -   (qqq) the nucleotide sequence of FIG. 6UU (MH74) (SEQ ID NO:85);    -   (rrr) the nucleotide sequence of FIG. 6WW (MH78) (SEQ ID NO:87);    -   (sss) the nucleotide sequence of FIG. 6YY (MH82) (SEQ ID NO:89);    -   (ttt) the nucleotide sequence of FIG. 6AAA (MH90) (SEQ ID        NO:91);    -   (uuu) the nucleotide sequence of FIG. 6CCC (MH94) (SEQ ID        NO:93);    -   (vvv) the nucleotide sequence of FIG. 6EEE (MH95) (SEQ ID        NO:95);    -   (www) the nucleotide sequence of FIG. 6GGG (MH107) (SEQ ID        NO:97);    -   (xxx) the nucleotide sequence of FIG. 6III (MH113) (SEQ ID        NO:99);    -   (yyy) the nucleotide sequence of FIG. 6KKK (MM4) (SEQ ID        NO:101);    -   (zzz) the nucleotide sequence of FIG. 6MMM (MM7) (SEQ ID        NO:103);    -   (aaaa) the nucleotide sequence of FIG. 6OOO (MM19) (SEQ ID        NO:105); (bbbb) the nucleotide sequence of FIG. 6QQQ (MM35) (SEQ        ID NO:107);    -   (cccc) the nucleotide sequence of FIG. 6SSS (MM44) (SEQ ID        NO:109);    -   (dddd) the nucleotide sequence of FIG. 6UUU (MM55) (SEQ ID        NO:111);    -   (eeee) the nucleotide sequence of FIG. 6WWW (MM65) (SEQ ID        NO:113);    -   (ffff) the nucleotide sequence of FIG. 6YYY (MM68) (SEQ ID        NO:115);    -   (gggg) the nucleotide sequence of 6AAAA (MM84) (SEQ ID NO:117);    -   (hhhh) the nucleotide sequence of FIG. 6CCCC (MM107) (SEQ ID        NO:119);    -   (iiii) the nucleotide sequence of FIG. 6EEEE (MM112) (SEQ ID        NO:121);    -   (jjjj) the nucleotide sequence of FIG. 6GGGG (MM115) (SEQ ID        NO:123);    -   (kkkk) the nucleotide sequence of FIG. 6IIII (MM120) (SEQ ID        NO:125);    -   (llll) the nucleotide sequence of FIG. 6KKKK (MM123) (SEQ ID        NO:127);    -   (mmmm) the nucleotide sequence of FIG. 6MMMM (MM136) (SEQ ID        NO:129);    -   (nnnn) the nucleotide sequence of FIG. 6OOOO (MM138) (SEQ ID        NO:131);    -   (oooo) the nucleotide sequence of FIG. 6QQQQ (MM141) (SEQ ID        NO:133);    -   (pppp) the nucleotide sequence of FIG. 6SSSS (MM144) (SEQ ID        NO:135),    -   (rrrr) or the nucleotide sequence of FIG. 6UUUU (MM153) (SEQ ID        NO:137); and    -   (rrrr) a nucleotide sequence that encodes an AAV capsid encoded        by the nucleotide sequence of any of (a) to (qqqq) but that        differs from the nucleotide sequences of (a) to (qqqq) due to        the degeneracy of the genetic code.

As an additional aspect, the invention provides AAV capsids encoded bythe nucleic acids of the invention.

The invention further provides viral particles comprising a virus genome(e.g., an AAV genome); and an AAV capsid of the invention, wherein theAAV capsid encapsidates the virus genome. In particular embodiments, thevirus genome is a recombinant vector genome comprising a heterologousnucleic acid.

Still further, the invention provides a pharmaceutical formulationcomprising a nucleic acid, AAV capsid or virus particle of the inventionin a pharmaceutically acceptable carrier.

As a further aspect, the invention provides a method of producing arecombinant virus particle comprising an AAV capsid (e.g., AAVparticle), the method comprising:

providing a cell in vitro with a nucleic acid of the present invention,an AAV rep coding sequence, a recombinant vector genome (e.g., a rAAVgenome) comprising a heterologous nucleic acid, and helper functions forgenerating a productive infection; and

allowing assembly of the recombinant virus particle comprising the AAVcapsid and encapsidating the recombinant vector genome.

The invention further provides methods of delivering a nucleic acid ofinterest to a cell, the method comprising administering a nucleic acid,virus particle or AAV capsid of the invention to the cell.

As still another aspect, the invention provides a method of delivering anucleic acid of interest to a mammalian subject, the method comprising:

administering an effective amount of a virus particle, nucleic acid,pharmaceutical formulation or AAV capsid of the invention to a mammaliansubject.

As yet another aspect, the invention provides a method of treatingmuscular dystrophy in a subject in need thereof, the method comprising:

administering an effective amount of a virus particle (e.g., an AAVparticle) or a pharmaceutical formulation comprising a virus particle ofthe invention to the mammalian subject, wherein the virus particlecomprises a heterologous nucleic acid encoding dystrophin, amini-dystrophin, a micro-dystrophin, a laminin-α2, a mini-agrin, anα-sarcoglycan, β-sarcoglycan, a γ-sarcoglycan, a δ-sarcoglycan,utrophin, Fukutin-related protein, myostatin pro-peptide, follistatin,dominant negative myostatin, an angiogenic factor and/or ananti-apoptotic factor.

Still further, the invention provides a method of treating congenitalheart failure in a subject in need thereof, the method comprising:

administering an effective amount of a virus particle (e.g., an AAVparticle) or pharmaceutical formulation comprising a virus particle ofthe invention to the mammalian subject, wherein the AAV particlecomprises a heterologous nucleic acid encoding a sarcoplasmicendoreticulum Ca²⁺-ATPase (SERCA2a), a β-adrenergic receptor kinaseinhibitor (βARKct), inhibitor 1 of protein phosphatase 1, ananti-apoptotic factor and/or an angiogenic factor.

The invention also encompasses a method of identifying a virus vector(e.g., an AAV vector) or AAV capsid having a tropism profile ofinterest, the method comprising:

-   -   (a) providing a collection of virus vectors (e.g., AAV vectors),        wherein each virus vector within the collection comprises:        -   (i) an AAV capsid comprising capsid proteins generated by            shuffling the capsid coding sequences of two or more            different AAV, wherein the capsid amino acid sequences of            the two or more different AAV differ by at least two amino            acids; and        -   (ii) a virus vector genome (e.g., an AAV vector genome)            comprising:            -   a cap coding sequence encoding the AAV capsid of (i);            -   an AAV rep coding sequence; and            -   at least one terminal repeat that functions with the Rep                protein(s) encoded by the AAV rep coding sequence (e.g.,                5′ and/or 3′ terminal repeats such as 5′ and/or 3′ AAV                terminal repeats);        -   wherein the AAV capsid encapsidates the virus vector genome;    -   (b) administering the collection of virus vectors to a mammalian        subject; and    -   (c) recovering a plurality of virus vectors as virions or as        viral genomes encoding the AAV capsid from a target tissue,        thereby identifying a virus vector or AAV capsid having a        tropism of interest.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Taqα I digested capsid genes of random clones from theplasmid library. The progeny capsid gene clones and parental capsid geneclones are indicated. Numbers 1-9 show capsid genotypes of thecorresponding AAV serotypes. “M” is the 100 base pair DNA size ladder.“R” designates recombinant.

FIG. 2 shows the frequencies of AAV clones retrieved from skeletalmuscle and liver after two rounds of in vivo panning. Forty three uniqueAAV mutants were identified from muscle after the first round ofscreening by their unique capsid sequences. Those infectious plasmidswere then mixed for production of a virus library for the second roundof biopanning in skeletal muscle after i.v. injection. Only thosemutants appearing more than once in the muscle are shown. Among the 79randomly retrieved mutants, 19 capsid sequences were found unique.

FIG. 3 depicts the structure and sequence of the mosaic capsid genes.The deduced M41 capsid amino acid (AA) sequence was aligned with that ofthe eight parental AAV serotypes to determine their phylogeneticrelationship (FIG. 3A). Different fill patterns of the sequence segmentsrepresent the AAV serotype of origin (i.e., 1, 6, 7 and 8). One residuereplacement distinct from any known AAV capsid (tyrosine 418) isindicated with a triangle. AA residue numbers begin from the start ofthe capsid open reading frame. FIG. 3B and FIG. 3C respectively depictthe nucleotide and amino acid sequence of the M41 capsid gene. FIGS.3D-3DD depict the structure, nucleotide and amino acid sequence ofmosaic capsid genes from vectors with skeletal muscle tropism, whereasFIGS. 3EE-3EEE depict the structure, nucleotide and amino acid sequenceof mosaic capsid genes from vectors with heart tropism.

FIG. 4 shows luciferase activities and vector genome copy numbers invarious mouse tissues after systemic administration of AAV vectors.Comparison of AAV9- and AAVM41-CMV-Luc vectors in luciferase activities(FIG. 4A), and AAV vector genome copy numbers (FIG. 4B), at 2-weeksafter intravenous injection of 3×10¹¹ vector genomes in mice. Similarcomparison of AAV6- or M41-CMV-Luc vectors in luciferase activities(FIG. 4C), and AAV genome copy numbers (FIG. 4D). Data are meanvalues±s.d. Ab, abdomen muscle; Di, diaphragm; Ta, tibialis anterior;Ga, gastrocnemius; Qd, quadriceps; Fl, forelimb; Ma, masseter; Ht,heart; Lv, liver; Sp, spleen; Kd, kidney; Lu, lung; Pa, pancreas; Te,testis; Br, brain; Tg, tongue. Heart vs. liver ratio in transductionefficiency by three rAAV vectors on luciferase activities (FIG. 4E), andvector genome copy numbers (FIG. 4F).

FIG. 5 shows the results of systemic delivery of LacZ transgene byAAVM41 into striated muscles. (FIG. 5A) X-gal staining of cross-sectionsof hearts after systemic administration of AAV vectors. 3×10¹¹ vectorgenomes of AAV9- or M41-CB-LacZ were injected via tail vein into adultmice; and 1×10¹² vector genomes of AAV9- or M41-CMV-LacZ were injectedvia jugular vein into adult hamsters. Hearts from mice or hamsters werecollected at 2-weeks or 3-weeks post injection for X-gal and eosinstaining. Two magnifications (4× and 40×) were used for photography.(FIG. 5B) LacZ transgene expression in the tibialis anterior muscles ofthe same animals as described in (A). Scale bars represented 200 μm (4×)and 50 μm (40×) in (A) and 100 μm in (B).

FIG. 6 depicts the nucleic acid and amino acid sequences of mosaic AAVcapsids following reshuffling, generation of secondary libraries, andscreening in vivo in heart and skeletal muscle tissue. The AAV capsidclones identified in Example 2 by screening heart tissue were reshuffledto generate a secondary heart library. Similarly, a secondary skeletalmuscle library was generated from the capsid mutants identified inExample 4 in skeletal muscle. The secondary heart library was subjectedto three successive screenings to identify those AAV capsid clonestargeting heart (“HH” designation; FIGS. 6H to 6Z). The secondaryskeletal muscle library was used for parallel screening for capsidclones targeting heart (designated “MH”; FIGS. 6AA to 6JJJ) and skeletalmuscle (designated “MM”; FIGS. 6KKK to 6VVVV).

FIG. 7 shows a comparison of gene transfer efficiency in primarycardiomyocytes or skeletal muscles. (FIG. 7A) Representative X-galstaining of cardiomyocytes or skeletal muscle after transduction byAAV9-, AAVM41- or AAV6-CMV-LacZ vectors. The AAV vectors were inoculatedon the primary neonatal rat cardiomyocytes (5×10⁵ cells/well) at aninfection multiplicity of 3000. Cells were fixed for X-gal staining 96hrs later (Top panels). The AAV vectors (5×10⁹ v.g.) were also injectedinto the gastrocnemius muscle of adult C57BJ/6L mice and tissues weresectioned and stained with X-gal and eosin 14 days post-treatment(bottom panels). Scale bars represent 100 μM. (FIGS. 7B, 7C)Quantitative β-gal activities and AAV vector genome copies in primarycardiomyocytes; and (FIG. 7D) f3-gal activities in mouse skeletalmuscles. Data are shown as mean values±s.d.

FIG. 8 depicts the results of studies of systemic delivery ofδ-sarcoglycan into cardiomyopathic hamster for treatment of heartfailure. 1×10¹² vector genomes of M41-Syn-BSG vector=were injected into7-week-old male TO-2 hamsters via the jugular vein (n=5). (FIG. 8A)Immunofluorescent staining of δ-sarcoglycan on thin sections of heartand skeletal muscle tissues 4 months after vector administration. Twomagnifications were used for clear view and the scale bars represent 50μn. (FIG. 8B) Western analysis of δ-sarcoglycan in muscle and non-muscletissues from untreated TO-2 (−) and rM41 vector-treated TO-2 (+)hamsters. 20 mg of total proteins were loaded in each lane. (FIG. 8C)Amelioration of cardiomyopathy in TO-2 hamsters after δ-sarcoglycan genetransfer. Hamster hearts of wild-type F1B, untreated TO-2 andAAVM41-δSG-treated TO-2 were cryosectioned and stained with differentmethods for histology and pathology. Arrows and arrowheads indicatedfibrosis and calcification respectively. Scale bars represent 100 μm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the inventors' discoverythat directed evolution and in vivo panning can be used to identifyscrambled AAV capsids and viruses comprising the same having desiredcharacteristics such as tropism profile and/or neutralization profile(e.g., ability to evade neutralizing antibodies) by scrambling AAVcapsid sequences from two or more different AAV capsids and thenscreening directly in vivo. In general, the methods of the invention arecarried out to identify novel AAV capsids comprising modifications inthe nucleic acid coding sequence(s) and/or amino acid sequence(s) forone, two or all three of the AAV capsid protein(s). The methods of theinvention based on in vivo screening address short-comings of prior cellculture based systems which are unable to simultaneously mimic in vivoconditions (e.g., the tight endothelial liming, differentiated skeletalmuscle cells, the effects of the liver on vector biodistribution, etc.).

As one illustrative example, the inventors have practiced the methods ofthe invention to identify in vivo AAV capsid mutants having a desiredtropism profile such as inefficient tropism for liver and/or efficienttropism for skeletal muscle (e.g., tongue muscle), diaphragm muscleand/or cardiac muscle.

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

Except as otherwise indicated, standard methods known to those skilledin the art may be used for production of recombinant and syntheticpolypeptides, antibodies or antigen-binding fragments thereof,manipulation of nucleic acid sequences, production of transformed cells,the construction of rAAV constructs, modified capsid proteins, packagingvectors expressing the AAV rep and/or cap sequences, and transiently andstably transfected packaging cells. Such techniques are known to thoseskilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: ALABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989); F. M.AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York).

All publications, patent applications, patents, nucleotide sequences,amino acid sequences and other references mentioned herein areincorporated by reference in their entirety.

I. DEFINITIONS

The designation of all amino acid positions in the AAV capsid subunitsin the description of the invention and the appended claims is withrespect to VP1 capsid subunit numbering.

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted.

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

The term “consisting essentially of” as used herein in connection with anucleic acid, protein or capsid structure means that the nucleic acid,protein or capsid structure does not contain any element other than therecited element(s) that significantly alters (e.g., more than about 1%,5% or 10%) the function of interest of the nucleic acid, protein orcapsid structure, e.g., tropism profile or neutralization profile of theprotein or capsid or a protein or capsid encoded by the nucleic acid.The term “adeno-associated virus” (AAV) in the context of the presentinvention includes without limitation AAV type 1, AAV type 2, AAV type 3(including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAVtype 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV,bovine AAV, canine AAV, equine AAV, and ovine AAV and any other AAV nowknown or later discovered. See, e.g., BERNARD N. FIELDS et al.,VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Anumber of additional AAV serotypes and clades have been identified (see,e.g., Gao et al., (2004) J. Virology 78:6381-6388 and Table 1), whichare also encompassed by the term “AAV.”

The genomic sequences of various AAV and autonomous parvoviruses, aswell as the sequences of the ITRs, Rep proteins, and capsid subunits areknown in the art. Such sequences may be found in the literature or inpublic databases such as the GenBank® database. See, e.g., GenBank®Accession Numbers NC 002077, NC 001401, NC 001729, NC 001863, NC 001829,NC 001862, NC 000883, NC 001701, NC 001510, AF063497, U89790, AF043303,AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962,AY028226, AY028223, NC 001358, NC 001540, AF513851, AF513852, AY530579,AY631965, AY631966; the disclosures of which are incorporated herein intheir entirety. See also, e.g., Srivistava et al., (1983) J. Virology45:555; Chiorini et al., (1998) J. Virology 71:6823; Chiorini et al.,(1999) J. Virology 73:1309; Bantel-Schaal et al., (1999) J. Virology73:939; Xiao et al., (1999) J. Virology 73:3994; Muramatsu et al.,(1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao etal., (2002) Proc. Nat. Acad. Sci. USA 99:11854; international patentpublications WO 00/28061, WO 99/61601, WO 98/11244; U.S. Pat. No.6,156,303; the disclosures of which are incorporated herein in theirentirety. See also Table 1. An early description of the AAV1, AAV2 andAAV3 terminal repeat sequences is provided by Xiao, X., (1996),“Characterization of

TABLE 1 GenBank Accession Complete Genomes Number Adeno-associated virus1 NC_002077, AF063497 Adeno-associated virus 2 NC_001401Adeno-associated virus 3 NC_001729 Adeno-associated virus NC_001863 3BAdeno-associated virus 4 NC_001829 Adeno-associated virus 5 Y18065,AF085716 Adeno-associated virus 6 NC_001862 Avian AAV ATCC VR- AY186198,AY629583, 865 NC_004828 Avian AAV strain DA-1 NC_006263, AY629583 BovineAAV NC_005889, AY388617 Clade A AAV1 NC_002077, AF063497 AAV6 NC_001862Hu.48 AY530611 Hu 43 AY530606 Hu 44 AY530607 Hu 46 AY530609 Clade B Hu.19 AY530584 Hu. 20 AY530586 Hu 23 AY530589 Hu22 AY530588 Hu24 AY530590Hu21 AY530587 Hu27 AY530592 Hu28 AY530593 Hu 29 AY530594 Hu63 AY530624Hu64 AY530625 Hu13 AY530578 Hu56 AY530618 Hu57 AY530619 Hu49 AY530612Hu58 AY530620 Hu34 AY530598 Hu35 AY530599 AAV2 NC_001401 Hu45 AY530608Hu47 AY530610 Hu51 AY530613 Hu52 AY530614 Hu T41 AY695378 Hu S17AY695376 Hu T88 AY695375 Hu T71 AY695374 Hu T70 AY695373 Hu T40 AY695372Hu T32 AY695371 Hu T17 AY695370 Hu LG15 AY695377 Clade C Hu9 AY530629Hu10 AY530576 Hu11 AY530577 Hu53 AY530615 Hu55 AY530617 Hu54 AY530616Hu7 AY530628 Hu18 AY530583 Hu15 AY530580 Hu16 AY530581 Hu25 AY530591Hu60 AY530622 Ch5 AY243021 Hu3 AY530595 Hu1 AY530575 Hu4 AY530602 Hu2AY530585 Hu61 AY530623 Clade D Rh62 AY530573 Rh48 AY530561 Rh54 AY530567Rh55 AY530568 Cy2 AY243020 AAV7 AF513851 Rh35 AY243000 Rh37 AY242998Rh36 AY242999 Cy6 AY243016 Cy4 AY243018 Cy3 AY243019 Cy5 AY243017 Rh13AY243013 Clade E Rh38 AY530558 Hu66 AY530626 Hu42 AY530605 Hu67 AY530627Hu40 AY530603 Hu41 AY530604 Hu37 AY530600 Rh40 AY530559 Rh2 AY243007 Bb1AY243023 Bb2 AY243022 Rh10 AY243015 Hu17 AY530582 Hu6 AY530621 Rh25AY530557 Pi2 AY530554 Pi1 AY530553 Pi3 AY530555 Rh57 AY530569 Rh50AY530563 Rh49 AY530562 Hu39 AY530601 Rh58 AY530570 Rh61 AY530572 Rh52AY530565 Rh53 AY530566 Rh51 AY530564 Rh64 AY530574 Rh43 AY530560 AAV8AF513852 Rh8 AY242997 Rh1 AY530556 Clade F Hu14 (AAV9) AY530579 Hu31AY530596 Hu32 AY530597 Clonal Isolate AAV5 Y18065, AF085716 AAV 3NC_001729 AAV 3B NC_001863 AAV4 NC_001829 Rh34 AY243001 Rh33 AY243002Rh32 AY243003Adeno-associated virus (AAV) DNA replication and integration,” Ph.D.Dissertation, University of Pittsburgh, Pittsburgh, Pa. (incorporatedherein it its entirety).

A “mosaic” or “scrambled” AAV nucleic acid capsid coding sequence or AAVcapsid protein is the result of scrambling or shuffling two or moredifferent AAV capsid sequences to produce capsid nucleic acid sequencesand amino acid sequences (i.e., AAV capsid proteins) that combineportions of two or more capsid sequences. A “mosaic” or “scrambled” AAVvirion or particle comprises a scrambled AAV capsid protein.

The term “tropism” as used herein refers to preferential entry of thevirus into certain cell or tissue type(s) and/or preferentialinteraction with the cell surface that facilitates entry into certaincell or tissue types, optionally and preferably followed by expression(e.g., transcription and, optionally, translation) of sequences carriedby the viral genome in the cell, e.g., for a recombinant virus,expression of the heterologous nucleotide sequence(s). Those skilled inthe art will appreciate that transcription of a heterologous nucleicacid sequence from the viral genome may not be initiated in the absenceof trans-acting factors, e.g., for an inducible promoter or otherwiseregulated nucleic acid sequence. In the case of a rAAV genome, geneexpression from the viral genome may be from a stably integratedprovirus and/or from a non-integrated episome, as well as any other formin which the virus nucleic acid may take within the cell.

The term “tropism profile” refers to the pattern of transduction of oneor more target cells, tissues and/or organs. For example, some scrambledAAV capsids may display efficient transduction of skeletal muscle (e.g.,tongue muscle), diaphragm muscle and/or cardiac muscle tissue.Conversely, some scrambled AAV capsids have only low level transductionof liver, gonads and/or germ cells. Representative examples of mosaic orscrambled AAV capsids have a tropism profile characterized by efficienttransduction of skeletal muscle, diaphragm muscle and/or cardiac musclewith only low transduction of liver.

As used herein, “transduction” of a cell by a virus vector (e.g., an AAVvector) means entry of the vector into the cell and transfer of geneticmaterial into the cell by the incorporation of nucleic acid into thevirus vector and subsequent transfer into the cell via the virus vector.

As used herein, a “collection” or “plurality” of virus particles,vectors, capsids or capsid proteins means two or more unless the contextindicates otherwise. Unless indicated otherwise, “efficienttransduction” or “efficient tropism,” or similar terms, can bedetermined by reference to a suitable control (e.g., at least about 50%,60%, 70%, 80%, 85%, 90%, 95% or more of the transduction or tropism,respectively, of the control). In particular embodiments, the virusvector efficiently transduces or has efficient tropism for skeletalmuscle, cardiac muscle, diaphragm muscle, pancreas (including a-isletcells), spleen, the gastrointestinal tract (e.g., epithelium and/orsmooth muscle), lung, joint cells, and/or kidney. Suitable controls willdepend on a variety of factors including the desired tropism profile.For example, AAV8 and AAV9 are highly efficient in transducing skeletalmuscle, cardiac muscle and diaphragm muscle, but have the disadvantageof also transducing liver with high efficiency. Thus, the invention canbe practiced to identify scrambled AAV that demonstrate the efficienttransduction of skeletal, cardiac and/or diaphragm muscle of AAV8 orAAV9, but with a much lower transduction efficiency for liver. Further,because the tropism profile of interest may reflect tropism towardmultiple target tissues, it will be appreciated that a suitablescrambled AAV may represent some tradeoffs. To illustrate, a scrambledAAV of the invention may be less efficient than AAV8 or AAV9 intransducing skeletal muscle, cardiac muscle and/or diaphragm muscle, butbecause of low level transduction of liver, may nonetheless be verydesirable.

Similarly, it can be determined if a virus “does not efficientlytransduce” or “does not have efficient tropism” for a target tissue, orsimilar terms, by reference to a suitable control. In particularembodiments, the virus vector does not efficiently, transduce (i.e., hasdoes not have efficient tropism) for liver, kidney, gonads and/or germcells. In particular embodiments, undesirable transduction of tissue(s)(e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1%or less of the level of transduction of the desired target tissue(s)(e.g., skeletal muscle, diaphragm muscle and/or cardiac muscle).

As used herein, the term “polypeptide” encompasses both peptides andproteins, unless indicated otherwise.

A “nucleic acid” or “nucleotide sequence” is a sequence of nucleotidebases, and may be RNA, DNA or DNA-RNA hybrid sequences (including bothnaturally occurring and non-naturally occurring nucleotide), but ispreferably either single or double stranded DNA sequences.

As used herein, an “isolated” nucleic acid or nucleotide sequence (e.g.,an “isolated DNA” or an “isolated RNA”) means a nucleic acid ornucleotide sequence separated or substantially free from at least someof the other components of the naturally occurring organism or virus,for example, the cell or viral structural components or otherpolypeptides or nucleic acids commonly found associated with the nucleicacid or nucleotide sequence.

Likewise, an “isolated” polypeptide means a polypeptide that isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other polypeptides or nucleicacids commonly found associated with the polypeptide.

By the term “treat,” “treating” or “treatment of” (or grammaticallyequivalent terms) it is meant that the severity of the subject'scondition is reduced or at least partially improved or amelioratedand/or that some alleviation, mitigation or decrease in at least oneclinical symptom is achieved and/or there is a delay in the progressionof the condition and/or prevention or delay of the onset of a disease ordisorder. The term “treat,” “treats,” “treating,” or “treatment of” andthe like also include prophylactic treatment of the subject (e.g., toprevent the onset of infection or cancer or a disorder). As used herein,the term “prevent,” “prevents,” or “prevention” (and grammaticalequivalents thereof) are not meant to imply complete abolition ofdisease and encompasses any type of prophylactic treatment that reducesthe incidence of the condition, delays the onset and/or progression ofthe condition, and/or reduces the symptoms associated with thecondition. Thus, unless the context indicates otherwise, the term“treat,” “treating” or “treatment of” (or grammatically equivalentterms) refer to both prophylactic and therapeutic regimens.

An “effective” amount as used herein is an amount that is sufficient toprovide some improvement or benefit to the subject. Alternativelystated, an “effective” amount is an amount that will provide somealleviation, mitigation, or decrease in at least one clinical symptom inthe subject. Those skilled in the art will appreciate that thetherapeutic effects need not be complete or curative, as long as somebenefit is provided to the subject.

A “heterologous nucleotide sequence” or “heterologous nucleic acid” istypically a sequence that is not naturally occurring in the virus.Generally, the heterologous nucleic acid or nucleotide sequencecomprises an open reading frame that encodes a polypeptide and/or anontranslated RNA.

A “therapeutic polypeptide” can be a polypeptide that can alleviate orreduce symptoms that result from an absence or defect in a protein in acell or subject. In addition, a “therapeutic polypeptide” can be apolypeptide that otherwise confers a benefit to a subject, e.g.,anti-cancer effects or improvement in transplant survivability.

As used herein, the term “vector,” “virus vector,” “delivery vector”(and similar terms) generally refers to a virus particle that functionsas a nucleic acid delivery vehicle, and which comprises the viralnucleic acid (i.e., the vector genome) packaged within the virion. Virusvectors according to the present invention comprise a scrambled AAVcapsid according to the invention and can package an AAV or rAAV genomeor any other nucleic acid including viral nucleic acids. Alternatively,in some contexts, the term “vector,” “virus vector,” “delivery vector”(and similar terms) may be used to refer to the vector genome (e.g.,vDNA) in the absence of the virion and/or to a viral capsid that acts asa transporter to deliver molecules tethered to the capsid or packagedwithin the capsid.

A “recombinant AAV vector genome” or “rAAV genome” is an AAV genome(i.e., vDNA) that comprises at least one inverted terminal repeat (e.g.,one, two or three inverted terminal repeats) and one or moreheterologous nucleotide sequences. rAAV vectors generally retain the 145base terminal repeat(s) (TR(s)) in cis to generate virus; however,modified AAV TRs and non-AAV TRs including partially or completelysynthetic sequences can also serve this purpose. All other viralsequences are dispensable and may be supplied in trans (Muzyczka, (1992)Curr. Topics Microbiol. Immunol. 158:97). The rAAV vector optionallycomprises two TRs (e.g., AAV TRs), which generally will be at the 5′ and3′ ends of the heterologous nucleotide sequence(s), but need not becontiguous thereto. The TRs can be the same or different from eachother. The vector genome can also contain a single ITR at its 3′ or 5′end.

The term “terminal repeat” or “TR” includes any viral terminal repeat orsynthetic sequence that forms a hairpin structure and functions as aninverted terminal repeat (i.e., mediates the desired functions such asreplication, virus packaging, integration and/or provirus rescue, andthe like). The TR can be an AAV TR or a non-AAV TR. For example, anon-AAV TR sequence such as those of other parvoviruses (e.g., canineparvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or theSV40 hairpin that serves as the origin of SV40 replication can be usedas a TR, which can further be modified by truncation, substitution,deletion, insertion and/or addition. Further, the TR can be partially orcompletely synthetic, such as the “double-D sequence” as described inU.S. Pat. No. 5,478,745 to Samulski et al.

An “AAV terminal repeat” or “AAV TR” may be from any AAV, including butnot limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or anyother AAV now known or later discovered (see, e.g., Table 1). An AAVterminal repeat need not have the native terminal repeat sequence (e.g.,a native AAV TR sequence may be altered by insertion, deletion,truncation and/or missense mutations), as long as the terminal repeatmediates the desired functions, e.g., replication, virus packaging,integration, and/or provirus rescue, and the like.

The terms “rAAV particle” and “rAAV virion” are used interchangeablyhere. A “rAAV particle” or “rAAV virion” comprises a rAAV vector genomepackaged within an AAV capsid.

The AAV capsid structure is described in more detail in BERNARD N.FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed.,Lippincott-Raven Publishers).

By “substantially retain” a property, it is meant that at least about75%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the property (e.g.,activity or other measurable characteristic) is retained.

II. SCRAMBLED AAV CAPSIDS IDENTIFIED BY DIRECTED EVOLUTION AND IN VIVOPANNING

The inventors have identified “scrambled” or “mosaic” AAV capsidstructures having characteristics of interest, e.g., tropism profileand/or neutralization profile. In particular embodiments, the scrambledcapsid demonstrates inefficient transduction of liver and/or efficienttransduction of skeletal muscle, diaphragm muscle and/or cardiac muscle.

Thus, in some embodiments, the invention provides scrambled AAV capsidscomprising, consisting of or consisting essentially of AAV capsidproteins having the amino acid sequences shown in FIGS. 3C, 3F, 3I, 3L,3O, 3R, 3U, 3S, 3AA, 3DD, 3GG, 3JJ, 3MM, 3PP, 3SS, 3VV, 3YY, 3BBB, 3EEE,6B, 6D, 6F, 6H, 6J, 6L, 6N, 6P, 6R, 6T, 6V, 6X, 6Z, 6BB, 6DD, 6FF, 6HH,6JJ, 6LL, 6NN, 6PP, 6RR, 6TT, 6VV, 6XX, 6ZZ, 6BBB, 6DDD, 6FFF, 6HHH,6JJJ, 6LLL, 6NNN, 6PPP, 6RRR, 6TTT, 6VVV, 6XXX, 6ZZZ, 6BBBB, 6DDDD,6FFFF, 6HHHH, 6JJJJ, 6LLLL, 6NNNN, 6PPPP, 6RRRR, 6TTTT and/or 6VVVV andviruses comprising the scrambled AAV capsids. These figures show the VP1capsid protein sequences. Those skilled in the art will understand thatthe AAV capsid generally contains the smaller VP2 and VP3 capsidproteins as well. Due to the overlap of the coding sequences for the AAVcapsid proteins, the nucleic acid coding sequences and amino acidsequences of the VP2 and VP3 capsid proteins will be apparent from theVP1 sequences shown in FIGS. 3A-3EEE and 6A-6VVVV.

In particular embodiments, the scrambled capsid protein can comprise,consist of, or consist essentially of the amino acid sequence of FIG. 3C(M41); the amino acid sequence of FIG. 3F (M17); the amino acid sequenceof FIG. 3I (M22); the amino acid sequence of FIG. 3L (M35); the aminoacid sequence of FIG. 3O (M42); the amino acid sequence of FIG. 3R(M62); the amino acid sequence of FIG. 3U (M67); the amino acid sequenceof FIG. 3X (M125); the amino acid sequence of FIG. 3AA (M148); the aminoacid sequence of FIG. 3DD (M151); the amino acid sequence of FIG. 3GG(H18); the amino acid sequence of FIG. 3JJ (H34); the amino acidsequence of FIG. 3MM (H39); the amino acid sequence of FIG. 3PP (H40);the amino acid sequence of FIG. 3SS(H43); the amino acid sequence ofFIG. 3VV (H50); the amino acid sequence of FIG. 3YY (H53); the aminoacid sequence of FIG. 3BBB (H66); the amino acid sequence of FIG. 3EEE(H109); the amino acid sequence of FIG. 6B (HH1); the amino acidsequence of FIG. 6D (HH15); the amino acid sequence of FIG. 6F (HH19);the amino acid sequence of FIG. 6H (HH27); the amino acid sequence ofFIG. 6J (HH35); the amino acid sequence of FIG. 6L (HH41); the aminoacid sequence of FIG. 6N(HH45); the amino acid sequence of FIG. 6P(HH53); the amino acid sequence of FIG. 6R (HH67); the amino acidsequence of FIG. 6T (HH68); the amino acid sequence of FIG. 6V (HH75);the amino acid sequence of FIG. 6X (HH87); the amino acid sequence ofFIG. 6Z (HH64); the amino acid sequence of FIG. 6BB (MH4); the aminoacid sequence of FIG. 6DD (MH18); the amino acid sequence of FIG. 6FF(MH21); the amino acid sequence of FIG. 6HH (MH31); the amino acidsequence of FIG. 6JJ (MH39); the amino acid sequence of FIG. 6LL(MHY43); the amino acid sequence of FIG. 6NN (MH47); the amino acidsequence of FIG. 6PP (MH58); the amino acid sequence of FIG. 6RR (MH63);the amino acid sequence of FIG. 6TT (MH71); the amino acid sequence ofFIG. 6VV (MH74); the amino acid sequence of FIG. 6XX (MH78); the aminoacid sequence of FIG. 6ZZ (MH82); the amino acid sequence of FIG. 6BBB(MH90); the amino acid sequence of FIG. 6DDD (MH94); the amino acidsequence of FIG. 6FFF (MH95); the amino acid sequence of FIG. 6HHH(MH107); the amino acid sequence of FIG. 6JJJ (MH113); the amino acidsequence of FIG. 6LLL (MM4); the amino acid sequence of FIG. 6NNN (MM7);the amino acid sequence of FIG. 6PPP (MM19); the amino acid sequence ofFIG. 6RRR (MM35); the amino acid sequence of FIG. 6TTT (MM44); the aminoacid sequence of FIG. 6VVV (MM55); the amino acid sequence of FIG. 6XXX(MM65); the amino acid sequence of FIG. 6ZZZ (MM68); the amino acidsequence of 6BBBB (MM84); the amino acid sequence of FIG. 6DDDD (MM107);the amino acid sequence of FIG. 6FFFF (MM112); the amino acid sequenceof FIG. 6HHHH (MM115); the amino acid sequence of FIG. 6JJJJ (MM120);the amino acid sequence of FIG. 6LLLL (MM123); the amino acid sequenceof FIG. 6NNNN (MM136); the amino acid sequence of FIG. 6PPPP (MM138);the amino acid sequence of FIG. 6RRRR (MM141); the amino acid sequenceof FIG. 6TTTT (MM144), or the amino acid sequence of FIG. 6VVVV (MM153).

Further, in non-limiting embodiments, the scrambled AAV capsids andcapsid proteins of the invention can be encoded by a nucleic acidcomprising, consisting of, or consisting essentially of the nucleotidesequence of FIG. 3E (M17); the nucleotide sequence of FIG. 3H (M22); thenucleotide sequence of FIG. 3K (M35); the nucleotide sequence of FIG. 3B(M41); the nucleotide sequence of FIG. 3N (M42); the nucleotide sequenceof FIG. 3Q (M62); the nucleotide sequence of FIG. 3T (M67); thenucleotide sequence of FIG. 3W (M125); the nucleotide sequence of FIG.3Z (M148); the nucleotide sequence of FIG. 3CC (M151); the nucleotidesequence of FIG. 3FF (H18); the nucleotide sequence of FIG. 3II (H34);the nucleotide sequence of FIG. 3LL (H39); the nucleotide sequence ofFIG. 3OO (H40); the nucleotide sequence of FIG. 3RR (H43); thenucleotide sequence of FIG. 3UU (H50); the nucleotide sequence of FIG.3XX (H53); the nucleotide sequence of FIG. 3AAA (H66); the nucleotidesequence of FIG. 3DDD (H109); the nucleotide sequence of FIG. 6A (HH1);the nucleotide sequence of FIG. 6C(HH15); the nucleotide sequence ofFIG. 6E (HH19); the nucleotide sequence of FIG. 6G (HH27); thenucleotide sequence of FIG. 6I (HH35); the nucleotide sequence of FIG.6K (HH41); the nucleotide sequence of FIG. 6M (HH45); the nucleotidesequence of FIG. 6O (HH53); the nucleotide sequence of FIG. 6Q (HH67);the nucleotide sequence of FIG. 6S(HH68); the nucleotide sequence ofFIG. 6U (HH75); the nucleotide sequence of FIG. 6W (HH87); thenucleotide sequence of FIG. 6Y (HH64); the nucleotide sequence of FIG.6AA (MH4); the nucleotide sequence of FIG. 6CC (MH18); the nucleotidesequence of FIG. 6EE (MH21); the nucleotide sequence of FIG. 6GG (MH31);the nucleotide sequence of FIG. 6II (MH39); the nucleotide sequence ofFIG. 6KK (MHY43); the nucleotide sequence of FIG. 6MM (MH47); thenucleotide sequence of FIG. 6OO (MH58); the nucleotide sequence of FIG.6QQ (MH63); the nucleotide sequence of FIG. 6SS (MH71); the nucleotidesequence of FIG. 6UU (MH74); the nucleotide sequence of FIG. 6WW (MH78);the nucleotide sequence of FIG. 6YY (MH82); the nucleotide sequence ofFIG. 6AAA (MH90); the nucleotide sequence of FIG. 6CCC (MH94); thenucleotide sequence of FIG. 6EEE (MH95); the nucleotide sequence of FIG.6GGG (MH107); the nucleotide sequence of FIG. 6III (MH113); thenucleotide sequence of FIG. 6KKK (MM4); the nucleotide sequence of FIG.6MMM (MM7); the nucleotide sequence of FIG. 6OOO (MM19); the nucleotidesequence of FIG. 6QQQ (MM35); the nucleotide sequence of FIG. 6SSS(MM44); the nucleotide sequence of FIG. 6OOO (MM55); the nucleotidesequence of FIG. 6WWW (MM65); the nucleotide sequence of FIG. 6YYY(MM68); the nucleotide sequence of 6AAAA (MM84); the nucleotide sequenceof FIG. 6CCCC (MM107); the nucleotide sequence of FIG. 6EEEE (MM112);the nucleotide sequence of FIG. 6GGGG (MM115); the nucleotide sequenceof FIG. 6IIII (MM120); the nucleotide sequence of FIG. 6KKKK (MM123);the nucleotide sequence of FIG. 6MMMM (MM136); the nucleotide sequenceof FIG. 6OOOO (MM138); the nucleotide sequence of FIG. 6QQQQ (MM141);the nucleotide sequence of FIG. 6SSSS (MM144), or the nucleotidesequence of FIG. 6UUUU (MM153); or a nucleotide sequence that encodes anAAV capsid or capsid protein encoded by the nucleotide sequence of anyof the foregoing but that differs from the nucleotide sequences of theforegoing due to the degeneracy of the genetic code.

The designation of all amino acid positions in the description of theinvention and the appended claims is with respect to VP1 numbering. Itwill be understood by those skilled in the art that due to the overlapin the AAV capsid coding sequences the modifications described hereincan also result in modifications in the VP2 and/or VP3 capsid subunits.

The invention also provides scrambled AAV capsid proteins and scrambledcapsids comprising, consisting of, or consisting essentially of thesame, wherein the capsid protein comprises, consists of, or consistsessentially of an amino acid sequence as shown in FIGS. 3C, 3F, 3I, 3L,3O, 3R, 3U, 3X, 3AA, 3DD, 3GG, 3JJ, 3MM, 3PP, 3SS, 3VV, 3YY, 3BBB, 3EEE,6B, 6D, 6F, 6H, 6J, 6L, 6N, 6P, 6R, 6T, 6V, 6X, 6Z, 6BB, 6DD, 6FF, 6HH,6JJ, 6LL, 6NN, 6PP, 6RR, 6TT, 6VV, 6XX, 6ZZ, 6BBB, 6DDD, 6FFF, 6HHH,6JJJ, 6LLL, 6NNNi, 6PPP, 6RRR, 6TTT, 6VVV, 6XXX, 6ZZZ, 6BBBB, 6DDDD,6FFFF, 6HHHH, 6JJJJ, 6LLLL, 6NNNN, 6PPPP, 6RRRR, 6TTTT and/or 6VVVV (anddescribed above), wherein 1, 2 or fewer, 3 or fewer, 4 or fewer, 5 orfewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 12or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 40 orfewer, or 50 or fewer of the amino acids within the capsid proteincoding sequence shown in FIGS. 3C, 3F, 3I, 3L, 30, 3R, 3U, 3X, 3AA, 3DD,3GG, 3JJ, 3MM, 3PP, 3SS, 3W, 3YY, 3BBB, 3EEE, 6B, 6D, 6F, 6H, 6J, 6L,6N, 6P, 6R, 6T, 6V, 6X, 6Z, 6BB, 6DD, 6FF, 6HH, 6JJ, 6LL, 6NN, 6PP, 6RR,6TT, 6VV, 6XX, 6ZZ, 6BBB, 6DDD, 6FFF, 6HHH, 6JJJ, 6LLL, 6NNN, 6PPP,6RRR, 6TTT, 6VVV, 6XXX, 6ZZZ, 6BBBB, 6DDDD, 6FFFF, 6HHHH, 6JJJJ, 6LLLL,6NNNN, 6PPPP, 6RRRR, 6TTTT and/or 6VVVV are substituted by another aminoacid (naturally occurring, modified and/or synthetic), optionally aconservative amino acid substitution, and/or are deleted and/or thereare insertions (including N-terminal and C-terminal extensions) of 1, 2or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8or fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15 or fewer, 20 orfewer, 25 or fewer, 30 or fewer, 40 or fewer, or 50 or fewer amino acidsor any combination of substitutions, deletions and/or insertions,wherein the substitutions, deletions and/or insertions do not undulyimpair the structure and/or function of a virion (e.g., an AAV virion)comprising the variant capsid protein or capsid. For example, inrepresentative embodiments of the invention, an AAV virion comprisingthe variant capsid protein substantially retains at least one propertyof a scrambled virion comprising a scrambled capsid protein as shown inFIGS. 3C, 3F, 3I, 3L, 3O, 3R, 3U, 3X, 3AA, 3DD, 3GG, 3JJ, 3MM, 3PP, 3SS,3W, 3YY, 3BBB, 3EEE, 6B, 6D, 6F, 6H, 6J, 6L, 6N, 6P, 6R, 6T, 6V, 6X, 6Z,6BB, 6DD, 6FF, 6HH, 6JJ, 6LL, 6NN, 6PP, 6RR, 6TT, 6VV, 6XX, 6ZZ, 6BBB,6DDD, 6FFF, 6HHH, 6JJJ, 6LLL, 6NNN, 6PPP, 6RRR, 6TTT, 6VVV, 6XXX, 6ZZZ,6BBBB, 6DDDD, 6FFFF, 6HHHH, 6JJJJ, 6LLLL, 6NNNN, 6PPPP, 6RRRR, 6TTTTand/or 6VVVV. For example, the virion comprising the variant capsidprotein can substantially retain the tropism profile of a virioncomprising the scrambled AAV capsid protein as shown in FIGS. 3C, 3F,3I, 3L, 3O, 3R, 3U, 3X, 3AA, 3DD, 3GG, 3JJ, 3MM, 3PP, 3SS, 3W, 3YY,3BBB, 3EEE, 6B, 6D, 6F, 6H, 6J, 6L, 6N, 6P, 6R, 6T, 6V, 6X, 6Z, 6BB,6DD, 6FF, 6HH, 6JJ, 6LL, 6NN, 6PP, 6RR, 6TT, 6VV, 6XX, 6ZZ, 6BBB, 6DDD,6FFF, 6HHH, 6JJJ, 6LLL, 6NNN, 6PPP, 6RRR, 6TTT, 6VVV, 6XXX, 6ZZZ, 6BBBB,6DDDD, 6FFFF, 6HHHH, 6JJJJ, 6LLLL, 6NNNN, 6PPPP, 6RRRR, 6TTTT and/or6VVVV (e.g., low efficiency transduction of liver and/or efficienttransduction of skeletal muscle, cardiac muscle, diaphragm muscle and/ortongue muscle). Methods of evaluating biological properties such asvirus transduction and/or neutralization by antibodies are well-known inthe art (see, e.g., the Examples).

Conservative amino acid substitutions are known in the art. Inparticular embodiments, a conservative amino acid substitution includessubstitutions within one or more of the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;asparagine, glutamine; serine, threonine; lysine, arginine; and/orphenylalanine, tyrosine.

It will be apparent to those skilled in the art that the amino acidsequences of the scrambled AAV capsid proteins in FIGS. 3C, 3F, 3I, 3L,3O, 3R, 3U, 3X, 3AA, 3DD, 3GG, 3JJ, 3MM, 3PP, 3SS, 3W, 3YY, 3BBB, 3EEE,6B, 6D, 6F, 6H, 6J, 6L, 6N, 6P, 6R, 6T, 6V, 6X, 6Z, 6BB, 6DD, 6FF, 6HH,6JJ, 6LL, 6NN, 6PP, 6RR, 6TT, 6VV, 6XX, 6ZZ, 6BBB, 6DDD, 6FFF, 6HHH,6JJJ, 6LLL, 6NNN, 6PPP, 6RRR, 6TTT, 6VVV, 6XXX, 6ZZZ, 6BBBB, 6DDDD,6FFFF, 6HHHH, 6JJJJ, 6LLLL, 6NNNN, 6PPPP, 6RRRR, 6TTTT and/or 6VVVV canfurther be modified to incorporate other modifications as known in theart to impart desired properties, for example, R484E and R585E mutationsto the AAV2 capsid sequence have been described that resulted inimproved cardiac transduction by AAV vector (Muller et al., (2006)Cardiovascular Research 70:70-78). As further nonlimiting possibilities,the capsid protein can be modified to incorporate targeting sequences(e.g., RGD) or sequences that facilitate purification and/or detection.For example, the capsid protein can be fused to all or a portion ofglutathione-S-transferase, maltose-binding protein, a heparin/heparansulfate binding domain, poly-His, a ligand, and/or a reporter protein(e.g., Green Fluorescent Protein, β-glucuronidase, β-galactosidase,luciferase, etc.), an immunoglobulin Fc fragment, a single-chainantibody, hemagglutinin, c-myc, FLAG epitope, and the like to form afusion protein. Methods of inserting targeting peptides into the AAVcapsid are known in the art (see, e.g., international patent publicationWO 00/28004; Nicklin et al., (2001) Molecular Therapy 474-181; White etal., (2004) Circulation 109:513-319; Muller et al., (2003) NatureBiotech. 21:1040-1046.

The viruses of the invention can further comprise a duplexed viralgenome as described in international patent publication WO 01/92551 andU.S. Pat. No. 7,465,583.

The invention also provides AAV capsids comprising the scrambled AAVcapsid proteins of the invention and virus particles (i.e., virions)comprising the same, wherein the virus particle packages (i.e.,encapsidates) a vector genome, optionally an AAV vector genome. Inparticular embodiments, the invention provides an AAV particlecomprising an AAV capsid comprising an AAV capsid protein of theinvention, wherein the AAV capsid packages an AAV vector genome. Theinvention also provides an AAV particle comprising an AAV capsid or AAVcapsid protein encoded by the scrambled nucleic acid capsid codingsequences of the invention. In particular embodiments, the virion is arecombinant vector comprising a heterologous nucleic acid of interest,e.g., for delivery to a cell. Thus, the present invention is useful forthe delivery of nucleic acids to cells in vitro, ex vivo, and in vivo.In representative embodiments, the recombinant vector of the inventioncan be advantageously employed to deliver or transfer nucleic acids toanimal (e.g., mammalian) cells.

Any heterologous nucleotide sequence(s) may be delivered by a virusvector of the present invention. Nucleic acids of interest includenucleic acids encoding polypeptides, optionally therapeutic (e.g., formedical or veterinary uses) and/or immunogenic (e.g., for vaccines)polypeptides.

Therapeutic polypeptides include, but are not limited to, cysticfibrosis transmembrane regulator protein (CFTR), dystrophin (includingthe protein product of dystrophin mini-genes or micro-genes, see, e.g,Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent PublicationNo. 2003017131; Wang et al., (2000) Proc Natl Acad Sci USA. 97:13714-9[mini-dystrophin]; Harper et al., (2002) Nat. Med. 8:253-61[micro-dystrophin]); mini-agrin, a laminin-α2, a sarcoglycan (α, β, γ orδ), Fukutin-related protein, myostatin pro-peptide, follistatin,dominant negative myostatin, an angiogenic factor (e.g., VEGF,angiopoietin-1 or 2), an anti-apoptotic factor (e.g., heme-oxygenase-1,TGF-β, inhibitors of pro-apoptotic signals such as caspases, proteases,kinases, death receptors [e.g., CD-095], modulators of cytochrome Crelease, inhibitors of mitochondrial pore opening and swelling); activintype II soluble receptor, anti-inflammatory polypeptides such as theIkappa B dominant mutant, sarcospan, utrophin, mini-utrophin, antibodiesor antibody fragments against myostatin or myostatin propeptide, cellcycle modulators, Rho kinase modulators such as Cethrin, which is amodified bacterial C3 exoenzyme [available from BioAxone Therapeutics,Inc., Saint-Lauren, Quebec, Canada], BCL-xL, BCL2, XIAP, FLICEc-s,dominant-negative caspase-8, dominant negative caspase-9, SPI-6 (see,e.g., U.S. Patent Application No. 20070026076), transcriptional factorPGC-α1, Pinch gene, ILK gene and thymosin β4 gene), clotting factors(e.g., Factor VIII, Factor IX, Factor X, etc.), erythropoietin,angiostatin, endostatin, catalase, tyrosine hydroxylase, anintracellular and/or extracellular superoxide dismutase, leptin, the LDLreceptor, neprilysin, lipoprotein lipase, ornithine transcarbamylase,β-globin, α-globin, spectrin, α₁-antitrypsin, adenosine deaminase,hypoxanthine guanine phosphoribosyl transferase, β-glucocerebrosidase,sphingomyelinase, lysosomal hexosaminidase A, branched-chain keto aciddehydrogenase, RP65 protein, a cytokine (e.g., α-interferon,βinterferon, interferon-γ, interleukins-1 through -14,granulocyte-macrophage colony stimulating factor, lymphotoxin, and thelike), peptide growth factors, neurotrophic factors and hormones (e.g.,somatotropin, insulin, insulin-like growth factors including IGF-1 andIGF-2, GLP-1, platelet derived growth factor, epidermal growth factor,fibroblast growth factor, nerve growth factor, neurotrophic factor-3 and-4, brain-derived neurotrophic factor, glial derived growth factor,transforming growth factor-α and β, and the like), bone morphogenicproteins (including RANKL and VEGF), a lysosomal protein, a glutamatereceptor, a lymphokine, soluble CD4, an Fc receptor, a T cell receptor,ApoE, ApoC, inhibitor 1 of protein phosphatase inhibitor 1 (I-1),phospholamban, serca2a, lysosomal acid α-glucosidase, α-galactosidase A,Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK),phosphoinositide-3 kinase (PI3 kinase), calsarcin, a receptor (e.g., thetumor necrosis growth factor-α soluble receptor), an anti-inflammatoryfactor such as IRAP, Pim-1, PGC-1α, SOD-1, SOD-2, ECF-SOD, kallikrein,thymosin-β4, hypoxia-inducible transcription factor [HIF], an angiogenicfactor, S100A1, parvalbumin, adenylyl cyclase type 6, a molecule thateffects G-protein coupled receptor kinase type 2 knockdown such as atruncated constitutively active bARKct; phospholamban inhibitory ordominant-negative molecules such as phospholamban S16E, a monoclonalantibody (including single chain monoclonal antibodies) or a suicidegene product (e.g., thymidine kinase, cytosine deaminase, diphtheriatoxin, and tumor necrosis factors such as TNF-α), and any otherpolypeptide that has a therapeutic effect in a subject in need thereof.

Heterologous nucleotide sequences encoding polypeptides include thoseencoding reporter polypeptides (e.g., an enzyme). Reporter polypeptidesare known in the art and include, but are not limited to, GreenFluorescent Protein, β-galactosidase, alkaline phosphatase, luciferase,and chloramphenicol acetyltransferase.

Alternatively, the heterologous nucleic acid may encode an antisenseoligonucleotide, a ribozyme (e.g., as described in U.S. Pat. No.5,877,022), RNAs that effect spliceosome-mediated trans-splicing (see,Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Pat. No.6,013,487; U.S. Pat. No. 6,083,702), interfering RNAs (RNAi) includingsmall interfering RNAs (siRNA) that mediate gene silencing (see, Sharpet al., (2000) Science 287:2431), microRNA, or other non-translated“functional” RNAs, such as “guide” RNAs (Gorman et al., (1998) Proc.Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.),and the like. Exemplary untranslated RNAs include RNAi or antisense RNAagainst the multiple drug resistance (MDR) gene product (e.g., to treattumors and/or for administration to the heart to prevent damage bychemotherapy), RNAi or antisense RNA against myostatin (Duchenne orBecker muscular dystrophy), RNAi or antisense RNA against VEGF or atumor immunogen including but not limited to those tumor immunogensspecifically described herein (to treat tumors), RNAi or antisenseoligonucleotides targeting mutated dystrophins (Duchenne or Beckermuscular dystrophy), RNAi or antisense RNA against the hepatitis Bsurface antigen gene (to prevent and/or treat hepatitis B infection),RNAi or antisense RNA against the HIV tat and/or rev genes (to preventand/or treat HIV) and/or RNAi or antisense RNA against any otherimmunogen from a pathogen (to protect a subject from the pathogen) or adefective gene product (to prevent or treat disease). RNAi or antisenseRNA against the targets described above or any other target can also beemployed as a research reagent.

As is known in the art, anti-sense nucleic acids (e.g., DNA or RNA) andinhibitory RNA (e.g., microRNA and RNAi such as siRNA or shRNA)sequences can be used to induce “exon skipping” in patients withmuscular dystrophy arising from defects in the dystrophin gene. Thus,the heterologous nucleic acid can encode an antisense nucleic acid orinhibitory RNA that induces appropriate exon skipping. Those skilled inthe art will appreciate that the particular approach to exon skippingdepends upon the nature of the underlying defect in the dystrophin gene,and numerous such strategies are known in the art. Exemplary antisensenucleic acids and inhibitory RNA sequences target the upstream branchpoint and/or downstream donor splice site and/or internal splicingenhancer sequence of one or more of the dystrophin exons (e.g., exons 19or 23). For example, in particular embodiments, the heterologous nucleicacid encodes an antisense nucleic acid or inhibitory RNA directedagainst the upstream branch point and downstream splice donor site ofexon 19 or 23 of the dystrophin gene. Such sequences can be incorporatedinto an AAV vector delivering a modified U7 snRNA and the antisensenucleic acid or inhibitory RNA (see, e.g., Goyenvalle et al., (2004)Science 306:1796-1799). As another strategy, a modified U1 snRNA can beincorporated into an AAV vector along with siRNA, microRNA or antisenseRNA complementary to the upstream and downstream splice sites of adystrophin exon (e.g., exon 19 or 23) (see, e.g., Denti et al. (2006)Proc. Nat. Acad. Sci. 103:3758-3763). Further, antisense nucleic acidsand inhibitory RNA can target the splicing enhancer sequences withinexons 19, 43, 45 or 53 (see, e.g., U.S. Pat. No. 6,653,467; U.S. Pat.No. 6,727,355; and U.S. Pat. No. 6,653,466).

Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim et al., (1987) Proc. Natl. Acad. Sci.USA 84:8788; Gerlach et al., (1987) Nature 328:802; Forster and Symons,(1987) Cell 49:211). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Michel and Westhof, (1990) J. Mol. Biol. 216:585;Reinhold-Hurek and Shub, (1992) Nature 357:173). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, (1989) Nature 338:217). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of nucleic acid expression may beparticularly suited to therapeutic applications (Scanlon et al., (1991)Proc. Natl. Acad. Sci. USA 88:10591; Sarver et al., (1990) Science247:1222; Sioud et al., (1992) J. Mol. Biol. 223:831).

MicroRNAs (mir) are natural cellular RNA molecules that can regulate theexpression of multiple genes by controlling the stability of the mRNA.Over-expression or diminution of a particular microRNA can be used totreat a dysfunction and has been shown to be effective in a number ofdisease states and animal models of disease (see, e.g., Couzin, (2008)Science 319:1782-4). The scrambled AAV can be used to deliver microRNAinto cells, tissues and subjects for the treatment of genetic andacquired diseases, or to enhance functionality and promote growth ofcertain tissues. For example, mir-1, mir-133, mir-206 and/or mir-208 canbe used to treat cardiac and skeletal muscle disease (see, e.g., Chen etal., (2006) Genet. 38:228-33; van Rooij et al., (2008) Trends Genet.24:159-66). MicroRNA can also be used to modulate the immune systemafter gene delivery (Brown et al., (2007) Blood 110:4144-52.)

The term “antisense oligonucleotide” (including “antisense RNA”) as usedherein, refers to a nucleic acid that is complementary to andspecifically hybridizes to a specified DNA or RNA sequence. Antisenseoligonucleotides and nucleic acids that encode the same can be made inaccordance with conventional techniques. See, e.g., U.S. Pat. No.5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et al.

Those skilled in the art will appreciate that it is not necessary thatthe antisense oligonucleotide be fully complementary to the targetsequence as long as the degree of sequence similarity is sufficient forthe antisense nucleotide sequence to specifically hybridize to itstarget (as defined above) and reduce production of the protein product(e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% ormore).

To determine the specificity of hybridization, hybridization of sucholigonucleotides to target sequences can be carried out under conditionsof reduced stringency, medium stringency or even stringent conditions.Suitable conditions for achieving reduced, medium and stringenthybridization conditions are as described herein.

Alternatively stated, in particular embodiments, antisenseoligonucleotides of the invention have at least about 60%, 70%, 80%,90%, 95%, 97%, 98% or higher sequence similarity with the complement ofthe target sequence and reduce production of the protein product (asdefined above). In some embodiments, the antisense sequence contains 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches as compared with the targetsequence.

Methods of determining percent identity of nucleic acid sequences aredescribed in more detail elsewhere herein.

The length of the antisense oligonucleotide is not critical as long asit specifically hybridizes to the intended target and reduces productionof the protein product (as defined above) and can be determined inaccordance with routine procedures. In general, the antisenseoligonucleotide is at least about eight, ten or twelve or fifteennucleotides in length and/or less than about 20, 30, 40, 50, 60, 70, 80,100 or 150 nucleotides in length.

An antisense oligonucleotide can be constructed using chemical synthesisand enzymatic ligation reactions by procedures known in the art. Forexample, an antisense oligonucleotide can be chemically synthesizedusing naturally occurring nucleotides or various modified nucleotidesdesigned to increase the biological stability of the molecules and/or toincrease the physical stability of the duplex formed between theantisense and sense nucleotide sequences, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used.

Examples of modified nucleotides which can be used to generate theantisense oligonucleotide include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopenten-yladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotides can further include nucleotide sequenceswherein at least one, or all, of the internucleotide bridging phosphateresidues are modified phosphates, such as methyl phosphonates, methylphosphonothioates, phosphoromorpholidates, phosphoropiperazidates andphosphoramidates. For example, every other one of the internucleotidebridging phosphate residues can be modified as described.

As another non-limiting example, one or all of the nucleotides in theoligonucleotide can contain a 2′ loweralkyl moiety (e.g., C₁-C₄, linearor branched, saturated or unsaturated alkyl, such as methyl, ethyl,ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example,every other one of the nucleotides can be modified as described. Seealso, Furdon et al., (1989) Nucleic Acids Res. 17, 9193-9204; Agrawal etal., (1990) Proc. Natl. Acad. Sci. USA 87, 1401-1405; Baker et al.,(1990) Nucleic Acids Res. 18, 3537-3543; Sproat et al., (1989) NucleicAcids Res. 17, 3373-3386; Walder and Walder, (1988) Proc. Natl. Acad.Sci. USA 85, 5011-5015.

The antisense oligonucleotide can be chemically modified (e.g., at the3′ and/or 5′ end) to be covalently conjugated to another molecule. Toillustrate, the antisense oligonucleotide can be conjugated to amolecule that facilitates delivery to a cell of interest, enhancesabsorption by the nasal mucosa (e.g, by conjugation to a lipophilicmoiety such as a fatty acid), provides a detectable marker, increasesthe bioavailability of the oligonucleotide, increases the stability ofthe oligonucleotide, improves the formulation or pharmacokineticcharacteristics, and the like. Examples of conjugated molecules includebut are not limited to cholesterol, lipids, polyamines, polyamides,polyesters, intercalators, reporter molecules, biotin, dyes,polyethylene glycol, human serum albumin, an enzyme, an antibody orantibody fragment, or a ligand for a cellular receptor.

Other modifications to nucleic acids to improve the stability,nuclease-resistance, bioavailabilityJormulation characteristics and/orpharmacokinetic properties are known in the art.

RNA interference (RNAi) is another useful approach for reducingproduction of a protein product (e.g., shRNA or siRNA). RNAi is amechanism of post-transcriptional gene silencing in whichdouble-stranded RNA (dsRNA) corresponding to a target sequence ofinterest is introduced into a cell or an organism, resulting indegradation of the corresponding mRNA. The mechanism by which RNAiachieves gene silencing has been reviewed in Sharp et al, (2001) GenesDev 15: 485-490; and Hammond et al., (2001) Nature Rev Gen 2:110-119).The RNAi effect persists for multiple cell divisions before geneexpression is regained. RNAi is therefore a powerful method for makingtargeted knockouts or “knockdowns” at the RNA level. RNAi has provensuccessful in human cells, including human embryonic kidney and HeLacells (see, e.g., Elbashir et al., Nature (2001) 411:494-8).

Initial attempts to use RNAi in mammalian cells resulted in antiviraldefense mechanisms involving PKR in response to the dsRNA molecules(see, e.g., Gil et al. (2000) Apoptosis 5:107). It has since beendemonstrated that short synthetic dsRNA of about 21 nucleotides, knownas “short interfering RNAs” (siRNA) can mediate silencing in mammaliancells without triggering the antiviral response (see, e.g., Elbashir etal., Nature (2001) 411:494-8; Caplen et al., (2001) Proc. Nat. Acad.Sci. 98:9742).

The RNAi molecule (including an siRNA molecule) can be a short hairpinRNA (shRNA; see Paddison et al., (2002), PNAS USA 99:1443-1448), whichis believed to be processed in the cell by the action of the RNase IIIlike enzyme Dicer into 20-25mer siRNA molecules. The shRNAs generallyhave a stem-loop structure in which two inverted repeat sequences areseparated by a short spacer sequence that loops out. There have beenreports of shRNAs with loops ranging from 3 to 23 nucleotides in length.The loop sequence is generally not critical. Exemplary loop sequencesinclude the following motifs: AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU,CCACACC and UUCAAGAGA.

The RNAi can further comprise a circular molecule comprising sense andantisense regions with two loop regions on either side to form a“dumbbell” shaped structure upon dsRNA formation between the sense andantisense regions. This molecule can be processed in vitro or in vivo torelease the dsRNA portion, e.g., a siRNA.

International patent publication WO 01/77350 describes a vector forbi-directional transcription to generate both sense and antisensetranscripts of a heterologous sequence in a eukaryotic cell. Thistechnique can be employed to produce RNAi for use according to theinvention.

Shinagawa et al. (2003) Genes & Dev. 17:1340 reported a method ofexpressing long dsRNAs from a CMV promoter (a pol II promoter), whichmethod is also applicable to tissue specific pol II promoters. Likewise,the approach of Xia et al., (2002) Nature Biotech. 20:1006, avoidspoly(A) tailing and can be used in connection with tissue-specificpromoters.

Methods of generating RNAi include chemical synthesis, in vitrotranscription, digestion of long dsRNA by Dicer (in vitro or in vivo),expression in vivo from a delivery vector, and expression in vivo from aPCR-derived RNAi expression cassette (see, e.g., TechNotes 10(3) “FiveWays to Produce siRNAs,” from Ambion, Inc., Austin Tex.; available atwww.ambion.com).

Guidelines for designing siRNA molecules are available (see e.g.,literature from Ambion, Inc., Austin Tex.; available at www.ambion.com).In particular embodiments, the siRNA sequence has about 30-50% G/Ccontent. Further, long stretches of greater than four T or A residuesare generally avoided if RNA polymerase III is used to transcribe theRNA. Online siRNA target finders are available, e.g., from Ambion, Inc.(www.ambion.com), through the Whitehead Institute of Biomedical Research(www.iura.wi.mit.edu) or from Dharmacon Research, Inc.(www.dharmacon.com/).

The antisense region of the RNAi molecule can be completelycomplementary to the target sequence, but need not be as long as itspecifically hybridizes to the target sequence (as defined above) andreduces production of the protein product (e.g., by at least about 30%,40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments,hybridization of such oligonucleotides to target sequences can becarried out under conditions of reduced stringency, medium stringency oreven stringent conditions, as defined above.

In other embodiments, the antisense region of the RNAi has at leastabout 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher sequence identity withthe complement of the target sequence and reduces production of theprotein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or more). In some embodiments, the antisense region contains 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches as compared with the targetsequence. Mismatches are generally tolerated better at the ends of thedsRNA than in the center portion. In particular embodiments, the RNAi isformed by intermolecular complexing between two separate sense andantisense molecules. The RNAi comprises a ds region formed by theintermolecular basepairing between the two separate strands. In'otherembodiments, the RNAi comprises a ds region formed by intramolecularbasepairing within a single nucleic acid molecule comprising both senseand antisense regions, typically as an inverted repeat (e.g., a shRNA orother stem loop structure, or a circular RNAi molecule). The RNAi canfurther comprise a spacer region between the sense and antisenseregions.

The RNAi molecule can contain modified sugars, nucleotides, backbonelinkages and other modifications as described above for antisenseoligonucleotides.

Generally, RNAi molecules are highly selective. If desired, thoseskilled in the art can readily eliminate candidate RNAi that are likelyto interfere with expression of nucleic acids other than the target bysearching relevant databases to identify RNAi sequences that do not havesubstantial sequence homology with other known sequences, for example,using BLAST (available at www.ncbi.nlm.nih.gov/BLAST).

Kits for the production of RNAi are commercially available, e.g., fromNew England Biolabs, Inc. and Ambion, Inc.

The recombinant virus vector may also comprise a heterologous nucleotidesequence that shares homology with and recombines with a locus on thehost chromosome. This approach may be utilized to correct a geneticdefect in the host cell.

The present invention also provides recombinant virus vectors thatexpress an immunogenic polypeptide, e.g., for vaccination. Theheterologous nucleic acid may encode any immunogen of interest known inthe art including, but are not limited to, immunogens from humanimmunodeficiency virus, influenza virus, gag proteins, tumor antigens,cancer antigens, bacterial antigens, viral antigens, and the like.Alternatively, the immunogen can be presented in the virus capsid (e.g.,incorporated therein) or tethered to the virus capsid (e.g., by covalentmodification).

The use of parvoviruses as vaccines is known in the art (see, e.g.,Miyamura et al., (1994) Proc. Nat. Acad. Sci. USA 91:8507; U.S. Pat. No.5,916,563 to Young et al., U.S. Pat. No. 5,905,040 to Mazzara et al.,U.S. Pat. No. 5,882,652, U.S. Pat. No. 5,863,541 to Samulski et al.; thedisclosures of which are incorporated herein in their entireties byreference). The antigen may be presented in the virus capsid.Alternatively, the antigen may be expressed from a heterologous nucleicacid introduced into a recombinant vector genome.

An immunogenic polypeptide, or immunogen, may be any polypeptidesuitable for protecting the subject against a disease, including but notlimited to microbial, bacterial, protozoal, parasitic, fungal and viraldiseases. For example, the immunogen may be an orthomyxovirus immunogen(e.g., an influenza virus immunogen, such as the influenza virushemagglutinin (HA) surface protein or the influenza virus nucleoproteingene, or an equine influenza virus immunogen), or a lentivirus immunogen(e.g., an equine infectious anemia virus immunogen, a SimianImmunodeficiency Virus (SIV) immunogen, or a Human ImmunodeficiencyVirus (HIV) immunogen, such as the HIV or SIV envelope GP160 protein,the HIV or SIV matrix/capsid proteins, and the HIV or SIV gag, pol andenv genes products). The immunogen may also be an arenavirus immunogen(e.g., Lassa fever virus immunogen, such as the Lassa fever virusnucleocapsid protein gene and the Lassa fever envelope glycoproteingene), a poxvirus immunogen (e.g., vaccinia, such as the vaccinia L1 orL8 genes), a flavivirus immunogen (e.g., a yellow fever virus immunogenor a Japanese encephalitis virus immunogen), a filovirus immunogen(e.g., an Ebola virus immunogen, or a Marburg virus immunogen, such asNP and GP genes), a bunyavirus immunogen (e.g., RVFV, CCHF, and SFSviruses), or a coronavirus immunogen (e.g., an infectious humancoronavirus immunogen, such as the human coronavirus envelopeglycoprotein gene, or a porcine transmissible gastroenteritis virusimmunogen, or an avian infectious bronchitis virus immunogen, or asevere acute respiratory syndrome (SARS) immunogen such as a S [S1 orS2], M, E, or N protein or an immunogenic fragment thereof). Theimmunogen may further be a polio immunogen, herpes immunogen (e.g., CMV,EBV, HSV immunogens) mumps immunogen, measles immunogen, rubellaimmunogen, diphtheria toxin or other diptheria immunogen, pertussisantigen, hepatitis (e.g., hepatitis A, hepatitis B or hepatitis C)immunogen, or any other vaccine immunogen known in the art.

Alternatively, the immunogen may be any tumor or cancer cell antigen.Optionally, the tumor or cancer antigen is expressed on the surface ofthe cancer cell. Exemplary cancer and tumor cell antigens are describedin S. A. Rosenberg, (1999) Immunity 10:281). Illustrative cancer andtumor antigens include, but are not limited to: BRCA1 gene product,BRCA2 gene product, gp100, tyrosinase, GAGE-1/2, BAGE, RAGE, NY-ESO-1,CDK-4, β-catenin, MUM-1, Caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15,melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad. Sci.USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami etal., (1994) Cancer Res. 54:3124) including MART-1 (Coulie et al., (1991)J. Exp. Med. 180:35), gp100 (Wick et al., (1988) J. Cutan. Pathol.4:201) and MAGE antigen (MAGE-1, MAGE-2 and MAGE-3) (Van der Bruggen etal., (1991) Science, 254:1643), CEA, TRP-1; TRP-2; P-15 and tyrosinase(Brichard et al., (1993) J. Exp. Med. 178:489); HER-2/neu gene product(U.S. Pat. No. 4,968,603); CA 125; HE4; LK26; FB5 (endosialin); TAG 72;AFP; CA19-9; NSE; DU-PAN-2; CA50; SPan-1; CA72-4; HCG; STN (sialyl Tnantigen); c-erbB-2 proteins; PSA; L-CanAg; estrogen receptor; milk fatglobulin; p53 tumor suppressor protein (Levine, (1993) Ann. Rev.Biochem. 62:623); mucin antigens (international patent publication WO90/05142); telomerases; nuclear matrix proteins; prostatic acidphosphatase; papilloma virus antigens; and antigens associated with thefollowing cancers: melanomas, adenocarcinoma, thymoma, sarcoma, lungcancer, liver cancer, colorectal cancer, non-Hodgkin's lymphoma,Hodgkin's lymphoma, leukemias, uterine cancer, breast cancer, prostatecancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer,pancreatic cancer, brain cancer, kidney cancer, stomach cancer,esophageal cancer, head and neck cancer and others (see, e.g.,Rosenberg, (1996) Ann. Rev. Med. 47:481-91).

Alternatively, the heterologous nucleotide sequence may encode anypolypeptide that is desirably produced in a cell in vitro, ex vivo, orin vivo. For example, the virus vectors may be introduced into culturedcells and the expressed protein product isolated therefrom.

It will be understood by those skilled in the art that the heterologousnucleic acid(s) of interest may be operably associated with appropriatecontrol sequences. For example, the heterologous nucleic acid may beoperably associated with expression control elements, such astranscription/translation control signals, origins of replication,polyadenylation signals, internal ribosome entry sites (IRES),promoters, enhancers, and the like.

Those skilled in the art will further appreciate that a variety ofpromoter/enhancer elements may be used depending on the level andtissue-specific expression desired. The promoter/enhancer may beconstitutive or inducible, depending on the pattern of expressiondesired. The promoter/enhancer may be native or foreign and can be anatural or a synthetic sequence. By foreign, it is intended that thetranscriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced.

Promoter/enhancer elements can be native to the target cell or subjectto be treated and/or native to the heterologous nucleic acid sequence.The promoter/enhancer element is generally chosen so that it willfunction in the target cell(s) of interest. In representativeembodiments, the promoter/enhancer element is a mammalianpromoter/enhancer element. The promoter/enhance element may beconstitutive or inducible.

Inducible expression control elements are generally used in thoseapplications in which it is desirable to provide regulation overexpression of the heterologous nucleic acid sequence(s). Induciblepromoters/enhancer elements for gene delivery can be tissue-specific ortissue-preferred promoter/enhancer elements, and include muscle specificor preferred (including cardiac, skeletal and/or smooth muscle), neuraltissue specific or preferred (including brain-specific), eye (includingretina-specific and cornea-specific), liver specific or preferred, bonemarrow specific or preferred, pancreatic specific or preferred, spleenspecific or preferred, and lung specific or preferred promoter/enhancerelements. Other inducible promoter/enhancer elements includehormone-inducible and metal-inducible elements. Exemplary induciblepromoters/enhancer elements include, but are not limited to, a Teton/off element, a RU486-inducible promoter, an ecdysone-induciblepromoter, a rapamycin-inducible promoter, and a metallothioneinpromoter.

In embodiments wherein which the heterologous nucleic acid sequence(s)is transcribed and then translated in the target cells, specificinitiation signals are generally employed for efficient translation ofinserted protein coding sequences. These exogenous translational controlsequences, which may include the ATG initiation codon and adjacentsequences, can be of a variety of origins, both natural and synthetic.

The invention also provides scrambled AAV particles comprising an AAVcapsid and an AAV genome, wherein the AAV genome “corresponds to” (i.e.,encodes) the AAV capsid. Also provided are collections or libraries ofsuch scrambled AAV particles, wherein the collection or librarycomprises 2 or more, 10 or more, 50 or more, 100 or more, 1000 or more,10⁴ or more, 10⁵ or more, or 10⁶ or more distinct sequences.

The present invention further encompasses “empty” capsid particles(i.e., in the absence of a vector genome) comprising, consisting of, orconsisting essentially of the scrambled AAV capsid proteins of theinvention. The scrambled AAV capsids of the invention can be used as“capsid vehicles,” as has been described in U.S. Pat. No. 5,863,541.Molecules that can be covalently linked, bound to or packaged by thevirus capsids and transferred into a cell include DNA, RNA, a lipid, acarbohydrate, a polypeptide, a small organic molecule, or combinationsof the same. Further, molecules can be associated with (e.g., “tetheredto”) the outside of the virus capsid for transfer of the molecules intohost target cells. In one embodiment of the invention the molecule iscovalently linked (i.e., conjugated or chemically coupled) to the capsidproteins. Methods of covalently linking molecules are known by thoseskilled in the art.

The virus capsids of the invention also find use in raising antibodiesagainst the novel capsid structures. As a further alternative, anexogenous amino acid sequence may be inserted into the virus capsid forantigen presentation to a cell, e.g., for administration to a subject toproduce an immune response to the exogenous amino acid sequence.

The invention also provides nucleic acids (e.g., isolated nucleic acids)encoding the scrambled virus capsids and scrambled capsid proteins ofthe invention. Further provided are vectors comprising the nucleicacids, and cells (in vivo or in culture) comprising the nucleic acidsand/or vectors of the invention. Such nucleic acids, vectors and cellscan be used, for example, as reagents (e.g., helper constructs orpackaging cells) for the production of virus vectors as describedherein.

In exemplary embodiments, the invention provides nucleic acid sequencesencoding the AAV capsids of FIGS. 3C, 3F, 3I, 3L, 30, 3R, 3U, 3X, 3ZZ,3DD, 3GG, 3JJ, 3MM, 3PP, 3SS, 3W, 3YY, 3BBB, 3EEE, 6B, 6D, 6F, 6H, 6J,6L, 6N, 6P, 6R, 6T, 6V, 6X, 6Z, 6BB, 6DD, 6FF, 6HH, 6JJ, 6LL, 6NN, 6PP,6RR, 6TT, 6VV, 6XX, 6ZZ, 6BBB, 6DDD, 6FFF, 6HHH, 6JJJ, 6LLL, 6NNN, 6PPP,6RRR, 6TTT, 6VVV, 6XXX, 6ZZZ, 6BBBB, 6DDDD, 6FFFF, 6HHHH, 6JJJJ, 6LLLL,6NNNN, 6PPPP, 6RRRR, 6TTTT and/or 6VVVV. Representative nucleic acidscomprise, consist of, or consist essentially of the sequences of FIG. 3,i.e., the nucleotide sequence of FIG. 3E (M17); the nucleotide sequenceof FIG. 3H (M22); the nucleotide sequence of FIG. 3K (M35); thenucleotide sequence of FIG. 3B (M41); the nucleotide sequence of FIG. 3N(M42); the nucleotide sequence of FIG. 3Q (M62); the nucleotide sequenceof FIG. 3T (M67); the nucleotide sequence of FIG. 3W (M125); thenucleotide sequence of FIG. 3Z (M148); the nucleotide sequence of FIG.3CC (M151); the nucleotide sequence of FIG. 3FF (H18); the nucleotidesequence of FIG. 3II (H34); the nucleotide sequence of FIG. 3LL (H39);the nucleotide sequence of FIG. 3OO (H40); the nucleotide sequence ofFIG. 3RR (H43); the nucleotide sequence of FIG. 3UU (H50); thenucleotide sequence of FIG. 3XX (H53); the nucleotide sequence of FIG.3AAA (H66); the nucleotide sequence of FIG. 3DDD (H109); the nucleotidesequence of FIG. 6A (HH1); the nucleotide sequence of FIG. 6C(HH15); thenucleotide sequence of FIG. 6E (HH19); the nucleotide sequence of FIG.6G (HH27); the nucleotide sequence of FIG. 6I (HH35); the nucleotidesequence of FIG. 6K (HH41); the nucleotide sequence of FIG. 6M (HH45);the nucleotide sequence of FIG. 6O (HH53); the nucleotide sequence ofFIG. 6Q (HH67); the nucleotide sequence of FIG. 6S (HH68); thenucleotide sequence of FIG. 6U (HH75); the nucleotide sequence of FIG.6W (HH87); the nucleotide sequence of FIG. 6Y (HH64); the nucleotidesequence of FIG. 6AA (MH4); the nucleotide sequence of FIG. 6CC (MH18);the nucleotide sequence of FIG. 6EE (MH21); the nucleotide sequence ofFIG. 6GG (MH31); the nucleotide sequence of FIG. 6II (MH39); thenucleotide sequence of FIG. 6KK (MHY43); the nucleotide sequence of FIG.6MM (MH47); the nucleotide sequence of FIG. 6OO (MH58); the nucleotidesequence of FIG. 6QQ (MH63); the nucleotide sequence of FIG. 6SS (MH71);the nucleotide sequence of FIG. 6UU (MH74); the nucleotide sequence ofFIG. 6WW (MH78); the nucleotide sequence of FIG. 6YY (MH82); thenucleotide sequence of FIG. 6AAA (MH90); the nucleotide sequence of FIG.6CCC (MH94); the nucleotide sequence of FIG. 6EEE (MH95); the nucleotidesequence of FIG. 6GGG (MH107); the nucleotide sequence of FIG. 6III(MH113); the nucleotide sequence of FIG. 6KKK (MM4); the nucleotidesequence of FIG. 6MMM (MM7); the nucleotide sequence of FIG. 6OOO(MM19); the nucleotide sequence of FIG. 6QQQ (MM35); the nucleotidesequence of FIG. 6SSS (MM44); the nucleotide sequence of FIG. 6OOO(MM55); the nucleotide sequence of FIG. 6WWW (MM65); the nucleotidesequence of FIG. 6YYY (MM68); the nucleotide sequence of 6AAAA (MM84);the nucleotide sequence of FIG. 6CCCC (MM107); the nucleotide sequenceof FIG. 6EEEE (MM112); the nucleotide sequence of FIG. 6GGGG (MM115);the nucleotide sequence of FIG. 6IIII (MM120); the nucleotide sequenceof FIG. 6KKKK (MM123); the nucleotide sequence of FIG. 6MMMM (MM136);the nucleotide sequence of FIG. 6OOOO (MM138); the nucleotide sequenceof FIG. 6QQQQ (MM141); the nucleotide sequence of FIG. 6SSSS (MM144), orthe nucleotide sequence of FIG. 6UUUU (MM153); or a nucleotide sequencethat encodes an AAV capsid encoded by the nucleotide sequence of any ofthe foregoing but that differs from the nucleotide sequences above dueto the degeneracy of the genetic code, which allows different nucleicacid sequences to code for the same polypeptide.

The invention also provides nucleic acids encoding the AAV capsidvariants, capsid protein variants and fusion proteins as describedabove. In particular embodiments, the nucleic acid hybridizes to thecomplement of the nucleic acid sequences specifically disclosed herein(e.g., see FIGS. 3B, 3E, 3H, 3K, 3N, 3Q, 3T, 3W, 3Z, 3CC, 3FF, 3II, 3LL,3OO, 3RR, 3UU, 3XX, 3AAA, 3DDD, 6A, 6C, 6E, 6G, 61, 6K, 6M, 6O, 6Q, 6S,6U, 6W, 6Y, 6AA, 6CC, 6EE, 6GG, 6II, 6KK, 6MM, 6OO, 6QQ, 6SS, 6UU, 6WW,6YY, 6AAA, 6CCC, 6EEE, 6GGG, 6III, 6KKK, 6MMM, 6OOO, 6QQQ, 6SSS, 6UUU,6WWW, 6YYY, 6AAAA, 6CCCC, 6EEEE, 6GGGG, 6IIII, 6KKKK, 6MMMM, 6OOOO,6QQQQ, 6SSSS and/or 6UUUU) under standard conditions as known by thoseskilled in the art and encodes a variant capsid and/or capsid protein.Optionally, the variant capsid or capsid protein substantially retainsat least one property of the capsid and/or capsid or capsid proteinencoded by the nucleic acid sequences of FIGS. 3B, 3E, 3H, 3K, 3N, 3Q,3T, 3W, 3Z, 3CC, 3FF, 3II, 3LL, 3OO, 3RR, 3UU, 3XX, 3AAA, 3DDD, 6A, 6C,6E, 6G, 6I, 6K, 6M, 6O, 6Q, 6S, 6U, 6W, 6Y, 6AA, 6CC, 6EE, 6GG, 6II,6KK, 6MM, 6OO, 6QQ, 6SS, 6UU, 6WW, 6YY, 6AAA, 6CCC, 6EEE, 6GGG, 6III,6KKK, 6MMM, 6OOO, 6QQQ, 6SSS, 6UUU, 6WWW, 6YYY, 6AAAA, 6CCCC, 6EEEE,6GGGG, 6IIII, 6KKKK, 6MMMM, 6OOOO, 6QQQQ, 6SSSS and/or 6UUUU. Forexample, a virus particle comprising the variant capsid or variantcapsid protein can substantially retain the tropism profile of a virusparticle comprising a capsid or capsid protein encoded by a nucleic acidcoding sequence as shown in FIGS. 3B, 3E, 3H, 3K, 3N, 3Q, 3T, 3W, 3Z,3CC, 3FF, 3II, 3LL, 3OO, 3RR, 3UU, 3XX, 3AAA, 3DDD, 6A, 6C, 6E, 6G, 6I,6K, 6M, 6O, 6Q, 6S, 6U, 6W, 6Y, 6AA, 6CC, 6EE, 6GG, 6II, 6KK, 6MM, 6OO,6QQ, 6SS, 6UU, 6WW, 6YY, 6AAA, 6CCC, 6EEE, 6GGG, 6III, 6KKK, 6MMM, 6OOO,6QQQ, 6SSS, 6UUU, 6WWW, 6YYY, 6AAAA, 6CCCC, 6EEEE, 6GGGG, 6IIII, 6KKKK,6MMMM, 6OOOO, 6QQQQ, 6SSSS and/or 6UUUU (e.g., inefficient transductionof liver and/or efficient transduction of skeletal muscle, cardiacmuscle and/or tongue muscle).

For example, hybridization of such sequences may be carried out underconditions of reduced stringency, medium stringency or even stringentconditions. Exemplary conditions for reduced, medium and stringenthybridization are as follows: (e.g., conditions represented by a washstringency of 35-40% Formamide with 5×Denhardt's solution, 0.5% SDS and1×SSPE at 37° C.; conditions represented by a wash stringency of 40-45%Formamide with 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.;and conditions represented by a wash stringency of 50% Formamide with5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively).See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual (2dEd. 1989) (Cold Spring Harbor Laboratory).

In other embodiments, nucleic acid sequences encoding a variant capsidor capsid protein of the invention have at least about 60%, 70%, 80%,85%, 90%, 95%, 97% or higher sequence identity with the nucleic acidsequences specifically disclosed in FIGS. 3B, 3E, 3H, 3K, 3N, 3Q, 3T,3W, 3Z, 3CC, 3FF, 3II, 3LL, 3OO, 3RR, 3UU, 3XX, 3AAA, 3DDD, 6A, 6C, 6E,6G, 6I, 6K, 6M, 6O, 6Q, 6S, 6U, 6W, 6Y, 6AA, 6CC, 6EE, 6GG, 6II, 6KK,6MM, 6OO, 6QQ, 6SS, 6UU, 6WW, 6YY, 6AAA, 6CCC, 6EEE, 6GGG, 6III, 6KKK,6MMM, 6OOO, 6QQQ, 6SSS, 6UUU, 6WWW, 6YYY, 6AAAA, 6CCCC, 6EEEE, 6GGGG,6IIII, 6KKKK, 6MMMM, 6OOOO, 6QQQQ, 6SSSS and/or 6UUUU) and optionallyencode a variant capsid or capsid protein that substantially retains atleast one property of the capsid or capsid protein encoded by a nucleicacid as shown in FIGS. 3B, 3E, 3H, 3K, 3N, 3Q, 3T, 3W, 3Z, 3CC, 3FF,3II, 3LL, 3OO, 3RR, 3UU, 3XX, 3AAA, 3DDD, 6A, 6C, 6E, 6G, 6I, 6K, 6M,6O, 6Q, 6S, 6U, 6W, 6Y, 6AA, 6CC, 6EE, 6GG, 6II, 6KK, 6MM, 6OO, 6QQ,6SS, 6UU, 6WW, 6YY, 6AAA, 6CCC, 6EEE, 6GGG, 6III, 6KKK, 6MMM, 6OOO,6QQQ, 6SSS, 6UUU, 6WWW, 6YYY, 6AAAA, 6CCCC, 6EEEE, 6GGGG, 6IIII, 6KKKK,6MMMM, 6OOOO, 6QQQQ, 6SSSS and/or 6UUUU.

As is known in the art, a number of different programs can be used toidentify whether a nucleic acid or polypeptide has sequence identity toa known sequence. Percent identity as used herein means that a nucleicacid or fragment thereof shares a specified percent identity to anothernucleic acid, when optimally aligned (with appropriate nucleotideinsertions or deletions) with the other nucleic acid (or itscomplementary strand), using BLASTN. To determine percent identitybetween two different nucleic acids, the percent identity is to bedetermined using the BLASTN program “BLAST 2 sequences”. This program isavailable for public use from the National Center for BiotechnologyInformation (NCBI) over the Internet (Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402). The parameters to be used are whatevercombination of the following yields the highest calculated percentidentity (as calculated below) with the default parameters shown inparentheses: Program—blastn Matrix—0 BLOSUM62 Reward for a match—0 or 1(1) Penalty for a mismatch—0, −1, −2 or −3 (−2) Open gap penalty—0, 1,2, 3, 4 or 5 (5) Extension gap penalty—0 or 1 (1) Gap x_dropoff—0 or 50(50) Expect—10.

Percent identity or similarity when referring to polypeptides, indicatesthat the polypeptide in question exhibits a specified percent identityor similarity when compared with another protein or a portion thereofover the common lengths as determined using BLASTP. This program is alsoavailable for public use from the National Center for BiotechnologyInformation (NCBI) over the Internet (Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402). Percent identity or similarity forpolypeptides is typically measured using sequence analysis software.See, e.g., the Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 910University Avenue, Madison, Wis. 53705. Protein analysis softwarematches similar sequences using measures of homology assigned to varioussubstitutions, deletions and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

In particular embodiments, the nucleic acid can comprise, consist of, orconsist essentially of a vector including but not limited to a plasmid,phage, viral vector (e.g., AAV vector, an adenovirus vector, aherpesvirus vector, or a baculovirus vector), bacterial artificialchromosome (BAC), or yeast artificial chromosome (YAC). For example, thenucleic acid can comprise, consist of, or consist essentially of an AAVvector comprising a 5′ and/or 3′ terminal repeat (e.g., 5′ and/or 3′ AAVterminal repeat).

In some embodiments the nucleic acid encoding the scrambled AAV capsidprotein further comprises an AAV Rep coding sequence. For example, thenucleic acid can be a helper construct for producing viral stocks.

The invention also provides packaging cells stably comprising a nucleicacid of the invention. For example, the nucleic acid can be stablyincorporated into the genome of the cell or can be stably maintained inan episomal form (e.g., an “EBV based nuclear episome”).

The nucleic acid can be incorporated into a delivery vector, such as aviral delivery vector. To illustrate, the nucleic acid of the inventioncan be packaged in an AAV particle, an adenovirus particle, aherpesvirus particle, a baculovirus particle, or any other suitablevirus particle.

Moreover, the nucleic acid can be operably associated with a promoterelement. Promoter elements are described in more detail herein.

The present invention further provides methods of producing the virusvectors of the invention. In a representative embodiment, the presentinvention provides a method of producing a recombinant virus vector, themethod comprising providing to a cell in vitro, (a) a templatecomprising (i) a heterologous nucleic acid, and (ii) packaging signalsequences sufficient for the encapsidation of the AAV template intovirus particles (e.g., one or more [e.g., two] terminal repeats, such asAAV terminal repeats), and (b) AAV sequences sufficient for replicationand encapsidation of the template into viral particles (e.g., the AAVrep and AAV cap sequences encoding an AAV capsid of the invention). Thetemplate and AAV replication and capsid sequences are provided underconditions such that recombinant virus particles comprising the templatepackaged within the capsid are produced in the cell. The method canfurther comprise the step of collecting the virus particles from thecell. Virus particles may be collected from the medium and/or by lysingthe cells.

In one illustrative embodiment, the invention provides a method ofproducing a rAAV particle comprising an AAV capsid, the methodcomprising: providing a cell in vitro with a nucleic acid encoding ascrambled AAV capsid of the invention, an AAV rep coding sequence, anAAV vector genome comprising a heterologous nucleic acid, and helperfunctions for generating a productive AAV infection; and allowingassembly of the AAV particles comprising the AAV capsid andencapsidating the AAV vector genome.

The cell is typically a cell that is permissive for AAV viralreplication. Any suitable cell known in the art may be employed, such asmammalian cells. Also suitable are trans-complementing packaging celllines that provide functions deleted from a replication-defective helpervirus, e.g., 293 cells or other E1a trans-complementing cells.

The AAV replication and capsid sequences may be provided by any methodknown in the art. Current protocols typically express the AAV rep/capgenes on a single plasmid. The AAV replication and packaging sequencesneed not be provided together, although it may be convenient to do so.The AAV rep and/or cap sequences may be provided by any viral ornon-viral vector. For example, the rep/cap sequences may be provided bya hybrid adenovirus or herpesvirus vector (e.g., inserted into the E1aor E3 regions of a deleted adenovirus vector). EBV vectors may also beemployed to express the AAV cap and rep genes. One advantage of thismethod is that EBV vectors are episomal, yet will maintain a high copynumber throughout successive cell divisions (i.e., are stably integratedinto the cell as extra-chromosomal elements, designated as an EBV basednuclear episome.

As a further alternative, the rep/cap sequences may be stably carried(episomal or integrated) within a cell.

Typically, the AAV rep/cap sequences will not be flanked by the AAVpackaging sequences (e.g., AAV ITRs), to prevent rescue and/or packagingof these sequences.

The template (e.g., an rAAV vector genome) can be provided to the cellusing any method known in the art. For example, the template may besupplied by a non-viral (e.g., plasmid) or viral vector. In particularembodiments, the template is supplied by a herpesvirus or adenovirusvector (e.g., inserted into the E1a or E3 regions of a deletedadenovirus). As another illustration, Palombo et al., (1998) J. Virology72:5025, describe a baculovirus vector carrying a reporter gene flankedby the AAV ITRs. EBV vectors may also be employed to deliver thetemplate, as described above with respect to the rep/cap genes.

In another representative embodiment, the template is provided by areplicating rAAV virus. In still other embodiments, an AAV provirus isstably integrated into the chromosome of the cell.

To obtain maximal virus titers, helper virus functions (e.g., adenovirusor herpesvirus) essential for a productive AAV infection are generallyprovided to the cell. Helper virus sequences necessary for AAVreplication are known in the art. Typically, these sequences areprovided by a helper adenovirus or herpesvirus vector. Alternatively,the adenovirus or herpesvirus sequences can be provided by anothernon-viral or viral vector, e.g., as a non-infectious adenovirusminiplasmid that carries all of the helper genes required for efficientAAV production as described by Ferrari et al., (1997) Nature. Med.3:1295, and U.S. Pat. Nos. 6,040,183 and 6,093,570.

Further, the helper virus functions nay be provided by a packaging cellwith the helper genes integrated in the chromosome or maintained as astable extrachromosomal element. In representative embodiments, thehelper virus sequences cannot be packaged into AAV virions, e.g., arenot flanked by AAV ITRs.

Those skilled in the art will appreciate that it may be advantageous toprovide the AAV replication and capsid sequences and the helper virussequences (e.g., adenovirus sequences) on a single helper construct.This helper construct may be a non-viral or viral construct, but isoptionally a hybrid adenovirus or hybrid herpesvirus comprising the AAVrep/cap genes.

In one particular embodiment, the AAV rep/cap sequences and theadenovirus helper sequences are supplied by a single adenovirus helpervector. This vector further contains the rAAV template. The AAV rep/capsequences and/or the rAAV template may be inserted into a deleted region(e.g., the E1a or E3 regions) of the adenovirus.

In a further embodiment, the AAV rep/cap sequences and the adenovirushelper sequences are supplied by a single adenovirus helper vector. TherAAV template is provided as a plasmid template.

In another illustrative embodiment, the AAV rep/cap sequences andadenovirus helper sequences are provided by a single adenovirus helpervector, and the rAAV template is integrated into the cell as a provirus.Alternatively, the rAAV template is provided by an EBV vector that ismaintained within the cell as an extrachromosomal element (e.g., as a“EBV based nuclear episome,” see Margolski, (1992) Curr. Top. Microbiol.Immun. 158:67).

In a further exemplary embodiment, the AAV rep/cap sequences andadenovirus helper sequences are provided by a single adenovirus helper.The rAAV template is provided as a separate replicating viral vector.For example, the rAAV template may be provided by a rAAV particle or asecond recombinant adenovirus particle.

According to the foregoing methods, the hybrid adenovirus vectortypically comprises the adenovirus 5′ and 3′ cis sequences sufficientfor adenovirus replication and packaging (i.e., the adenovirus terminalrepeats and PAC sequence). The AAV rep/cap sequences and, if present,the rAAV template are embedded in the adenovirus backbone and areflanked by the 5′ and 3′ cis sequences, so that these sequences may bepackaged into adenovirus capsids. As described above, in representativeembodiments, the adenovirus helper sequences and the AAV rep/capsequences are not flanked by the AAV packaging sequences (e.g., the AAVITRs), so that these sequences are not packaged into the AAV virions.

Herpesvirus may also be used as a helper virus in AAV packaging methods.Hybrid herpesviruses encoding the AAV Rep protein(s) may advantageouslyfacilitate for more scalable AAV vector production schemes. A hybridherpes simplex virus type I (HSV-1) vector expressing the AAV-2 rep andcap genes has been described (Conway et al., (1999) Gene Therapy 6:986and WO 00/17377, the disclosures of which are incorporated herein intheir entireties).

As a further alternative, the virus vectors of the invention can beproduced in insect cells using baculovirus vectors to deliver therep/cap genes and rAAV template as described by Urabe et al., (2002)Human Gene Therapy 13:1935-43.

Other methods of producing AAV use stably transformed packaging cells(see, e.g., U.S. Pat. No. 5,658,785).

AAV vector stocks free of contaminating helper virus may be obtained byany method known in the art. For example, AAV and helper virus may bereadily differentiated based on size. AAV may also be separated awayfrom helper virus based on affinity for a heparin substrate (Zolotukhinet al. (1999) Gene Therapy 6:973). In representative embodiments,deleted replication-defective helper viruses are used so that anycontaminating helper virus is not replication competent. As a furtheralternative, an adenovirus helper lacking late gene expression may beemployed, as only adenovirus early gene expression is required tomediate packaging of AAV virus. Adenovirus mutants defective for lategene expression are known in the art (e.g., ts100K and ts149 adenovirusmutants).

The inventive packaging methods may be employed to produce high titerstocks of virus particles. In particular embodiments, the virus stockhas a titer of at least about 10⁵ transducing units (tu)/ml, at leastabout 10⁶ tu/ml, at least about 10⁷ tu/ml, at least about 10⁸ tu/ml, atleast about 10⁹ tu/ml, or at least about 10¹⁰ tu/ml.

The novel capsid protein and capsid structures find use in raisingantibodies, for example, for diagnostic or therapeutic uses or as aresearch reagent. Thus, the invention also provides antibodies againstthe novel capsid proteins and capsids of the invention.

The term “antibody” or “antibodies” as used herein refers to all typesof immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodycan be monoclonal or polyclonal and can be of any species of origin,including (for example) mouse, rat, rabbit, horse, goat, sheep or human,or can be a chimeric antibody. See, e.g., Walker et al., Molec. Immunol.26, 403-11 (1989). The antibodies can be recombinant monoclonalantibodies, for example, produced according to the methods disclosed inU.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibodies canalso be chemically constructed, for example, according to the methoddisclosed in U.S. Pat. No. 4,676,980.

Antibody fragments included within the scope of the present inventioninclude, for example, Fab, F(ab′)2, and Fc fragments, and thecorresponding fragments obtained from antibodies other than IgG. Suchfragments can be produced by known techniques. For example, F(ab′)2fragments can be produced by pepsin digestion of the antibody molecule,and Fab fragments can be generated by reducing the disulfide bridges ofthe F(ab′)2 fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse et al., (1989) Science 254,1275-1281).

Polyclonal antibodies can be produced by immunizing a suitable animal(e.g., rabbit, goat, etc.) with an antigen to which a monoclonalantibody to the target binds, collecting immune serum from the animal,and separating the polyclonal antibodies from the immune serum, inaccordance with known procedures.

Monoclonal antibodies can be produced in a hybridoma cell line accordingto the technique of Kohler and Milstein, (1975) Nature 265, 495-97. Forexample, a solution containing the appropriate antigen can be injectedinto a mouse and, after a sufficient time, the mouse sacrificed andspleen cells obtained. The spleen cells are then immortalized by fusingthem with myeloma cells or with lymphoma cells, typically in thepresence of polyethylene glycol, to produce hybridoma cells. Thehybridoma cells are then grown in a suitable medium and the supernatantscreened for monoclonal antibodies having the desired specificity.Monoclonal Fab fragments can be produced in E. coli by recombinanttechniques known to those skilled in the art. See, e.g., W. Huse, (1989)Science 246, 1275-81.

Antibodies specific to a target polypeptide can also be obtained byphage display techniques known in the art.

Various immunoassays can be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificity are well known in theart. Such immunoassays typically involve the measurement of complexformation between an antigen and its specific antibody (e.g.,antigen/antibody complex formation). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes can be used as well as a competitive bindingassay.

Antibodies can be conjugated to a solid support (e.g., beads, plates,slides or wells formed from materials such as latex or polystyrene) inaccordance with known techniques. Antibodies can likewise be conjugatedto detectable groups such as radiolabels (e.g. ³⁵S, ¹²⁵I, ¹³¹I), enzymelabels (e.g., horseradish peroxidase, alkaline phosphatase), andfluorescence labels (e.g., fluorescein) in accordance with knowntechniques. Determination of the formation of an antibody/antigencomplex in the methods of this invention can be by detection of, forexample, precipitation, agglutination, flocculation, radioactivity,color development or change, fluorescence, luminescence, etc., as iswell known in the art.

The present invention also encompasses methods for deliveringheterologous nucleotide sequences into a broad range of cells, includingdividing and non-dividing cells. The virus vectors of the invention maybe employed to deliver a nucleotide sequence of interest to a cell invitro, e.g., to produce a polypeptide in vitro or for ex vivo genetherapy. The vectors are additionally useful in a method of delivering anucleotide sequence to a subject in need thereof, e.g., to express animmunogenic or therapeutic polypeptide. In this manner, the polypeptidemay thus be produced in vivo in the subject. The subject may be in needof the polypeptide because the subject has a deficiency of thepolypeptide, or because the production of the polypeptide in the subjectmay impart some therapeutic effect, as a method of treatment orotherwise, and as explained further below.

In general, the virus vectors of the invention may be employed todeliver any foreign nucleic acid with a biological effect to treat orameliorate the symptoms associated with any disorder related to geneexpression. Further, the invention can be used to treat any diseasestate for which it is beneficial to deliver a therapeutic polypeptide.Illustrative disease states include, but are not limited to: cysticfibrosis (cystic fibrosis transmembrane regulator protein) and otherdiseases of the lung, hemophilia A (Factor VIII), hemophilia B (FactorIX), thalassemia (γ-globin), anemia (erythropoietin) and other blooddisorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis(γ-interferon), Parkinson's disease (glial-cell line derivedneurotrophic factor [GDNF]), Huntington's disease (inhibitory RNAincluding without limitation RNAi such as siRNA or shRNA, antisense RNAor microRNA to remove repeats), amyotrophic lateral sclerosis, epilepsy(galanin, neurotrophic factors), and other neurological disorders,cancer (endostatin, angiostatin, TRAIL, FAS-ligand, cytokines includinginterferons; inhibitory RNA including without limitation RNAi (such assiRNA or shRNA), antisense RNA and microRNA including inhibitory RNAagainst VEGF, the multiple drug resistance gene product or a cancerimmunogen), diabetes mellitus (insulin, PGC-α1, GLP-1, myostatinpro-peptide, glucose transporter 4), muscular dystrophies includingDuchenne and Becker (e.g., dystrophin, mini-dystrophin,micro-dystrophin, insulin-like growth factor I, a sarcoglycan [e.g., α,β, γ], Inhibitory RNA [e.g., RNAi, antisense RNA or microRNA] againstmyostatin or myostatin propeptide, laminin-alpha2, Fukutan-relatedprotein, dominant negative myostatin, follistatin, activin type IIsoluble receptor, anti-inflammatory polypeptides such as the Ikappa Bdominant mutant, sarcospan, utrophin, mini-utrophin, inhibitory RNA[e.g., RNAi, antisense RNA or microRNA] against splice junctions in thedystrophin gene to induce exon skipping [see, e.g., WO/2003/095647],inhibitory RNA (e.g., RNAi, antisense RNA or micro RNA] against U7snRNAs to induce exon skipping [see, e.g., WO/2006/021724], andantibodies or antibody fragments against myostatin or myostatinpropeptide), Gaucher disease (glucocerebrosidase), Hurler's disease(α-L-iduronidase), adenosine deaminase deficiency (adenosine deaminase),glycogen storage diseases (e.g., Fabry disease [α-galactosidase] andPompe disease [lysosomal acid α-glucosidase]) and other metabolicdefects including other lysosomal storage disorders and glycogen storagedisorders, congenital emphysema (α1-antitrypsin), Lesch-Nyhan Syndrome(hypoxanthine guanine phosphoribosyl transferase), Niemann-Pick disease(sphingomyelinase), Maple Syrup Urine Disease (branched-chain keto aciddehydrogenase), retinal degenerative diseases (and other diseases of theeye and retina; e.g., PDGF, endostatin and/or angiostatin for maculardegeneration), diseases of solid organs such as brain (includingParkinson's Disease [GDNF], astrocytomas [endostatin, angiostatin and/orRNAi against VEGF], glioblastomas [endostatin, angiostatin and/or RNAiagainst VEGF]), liver (RNAi such as siRNA or shRNA, microRNA orantisense RNA for hepatitis B and/or hepatitis C genes), kidney, heartincluding congestive heart failure or peripheral artery disease (PAD)(e.g., by delivering protein phosphatase inhibitor I [I-1],phospholamban, sarcoplasmic endoreticulum Ca²⁺-ATPase [serca2a], zincfinger proteins that regulate the phospholamban gene, Pim-1, PGC-1α,SOD-1, SOD-2, ECF—SOD, kallikrein, thymosin-β4, hypoxia-inducibletranscription factor [HIF], βarkct, β2-adrenergic receptor,β2-adrenergic receptor kinase [PARK], phosphoinositide-3 kinase [PI3kinase], calsarcin, an angiogenic factor, S100A1, parvalbumin, adenylylcyclase type 6, a molecule that effects G-protein coupled receptorkinase type 2 knockdown such as a truncated constitutively activebARKct, an inhibitory RNA [e.g., RNAi, antisense RNA or microRNA]against phospholamban; phospholamban inhibitory or dominant-negativemolecules such as phospholamban S16E, etc.), arthritis (insulin-likegrowth factors), joint disorders (insulin-like growth factors), intimalhyperplasia (e.g., by delivering enos, inos), improve survival of hearttransplants (superoxide dismutase), AIDS (soluble CD4), muscle wasting(insulin-like growth factor I, myostatin pro-peptide, an anti-apoptoticfactor, follistatin), limb ischemia (VEGF, FGF, PGC-1α, EC-SOD, HIF),kidney deficiency (erythropoietin), anemia (erythropoietin), arthritis(anti-inflammatory factors such as IRAP and TNFα soluble receptor),hepatitis (α-interferon), LDL receptor deficiency (LDL receptor),hyperammonemia (ornithine transcarbamylase), spinal cerebral ataxiasincluding SCA1, SCA2 and SCA3, phenylketonuria (phenylalaninehydroxylase), autoimmune diseases, and the like. The invention canfurther be used following organ transplantation to increase the successof the transplant and/or to reduce the negative side effects of organtransplantation or adjunct therapies (e.g., by administeringimmunosuppressant agents or inhibitory nucleic acids to block cytokineproduction). As another example, bone morphogenic proteins (includingRANKL and/or VEGF) can be administered with a bone allograft, forexample, following a break or surgical removal in a cancer patient.

Exemplary lysosomal storage diseases that can be treated according tothe present invention include without limitation: Hurler's Syndrome (MPSIH), Scheie's Syndrome (MPS IS), and Hurler-Scheie Syndrome (MPS IH/S)(α-L-iduronidase); Hunter's Syndrome (MPS II) (iduronate sulfatesulfatase); Sanfilippo A Syndrome (MPS IIIA) (Heparan-S-sulfatesulfaminidase), Sanfilippo B Syndrome (MPS IIIB)(N-acetyl-D-glucosaminidase), Sanfilippo C Syndrome (MPS IIIC)(Acetyl-CoA-glucosaminide N-acetyltransferase), Sanfilippo D Syndrome(MPS IIID) (N-acetyl-glucosaminine-6-sulfate sulfatase); Morquio Adisease (MPS IVA) (Galactosamine-6-sulfate sulfatase), Morquio B disease(MPS IV B) (β-Galactosidase); Maroteaux-Imay disease (MPS VI)(arylsulfatase B); Sly Syndrome (MPS VII) (β-glucuronidase);hyaluronidase deficiency (MPS IX) (hyaluronidase); sialidosis(mucolipidosis I), mucolipidosis II (I-Cell disease)(N-actylglucos-aminyl-1-phosphotransferase catalytic subunit),mucolipidosis III (pseudo-Hurler polydystrophy)(N-acetylglucos-aminyl-1-phosphotransferase; type IIIA [catalyticsubunit] and type IIIC [substrate recognition subunit]); GM1gangliosidosis (ganglioside p-galactosidase), GM2 gangliosidosis Type I(Tay-Sachs disease) (β-hexaminidase A), GM2 gangliosidosis type II(Sandhoff's disease) (β-hexosaminidase B); Niemann-Pick disease (Types Aand B) (sphingomyelinase); Gaucher's disease (glucocerebrosidase);Farber's disease (ceraminidase); Fabry's disease (a-galactosidase A);Krabbe's disease (galactosylceramide β-galactosidase); metachromaticleukodystrophy (arylsulfatase A); lysosomal acid lipase deficiencyincluding Wolman's disease (lysosomal acid lipase); Batten disease(juvenile neuronal ceroid lipofuscinosis) (lysosomal trans-membrane CLN3protein) sialidosis (neuraminidase 1); galactosialidosis (Goldberg'ssyndrome) (protective protein/cathepsin A); α-mannosidosis(α-D-mannosidase); β-mannosidosis (β-D-mannosidosis); fucosidosis(α-D-fucosidase); aspartylglucosaminuria (N-Aspartylglucosaminidase);and sialuria (Na phosphate cotransporter).

Exemplary glycogen storage diseases that can be treated according to thepresent invention include, but are not limited to, Type Ia GSD (vonGierke disease) (glucose-6-phosphatase), Type Ib GSD (glucose-6-phophatetranslocase), Type Ic GSD (microsomal phosphate or pyrophosphatetransporter), Type Id GSD (microsomal glucose transporter), Type II GSDincluding Pompe disease or infantile Type IIa GSD (lysosomal acidα-glucosidase) and Type IIb (Danon) (lysosomal membrane protein-2), TypeIIIa and IIIb GSD (Debrancher enzyme; amyloglucosidase andoligoglucanotransferase), Type IV GSD (Andersen's disease) (branchingenzyme), Type V GSD (McArdle disease) (muscle phosphorylase), Type VIGSD (Hers' disease) (liver phosphorylase), Type VII GSD (Tarui'sdisease) (phosphofructokinase), GSD Type VIII/IXa (X-linkedphosphorylase kinase), GSD Type IXb (Liver and muscle phosphorylasekinase), GSD Type IXc (liver phosphorylase kinase), GSD Type IXd (musclephosphorylase kinase), GSD O (glycogen synthase), Fanconi-Bickelsyndrome (glucose transporter-2), phosphoglucoisomerase deficiency,muscle phosphoglycerate kinase deficiency, phosphoglycerate mutasedeficiency, fructose 1,6-diphosphatase deficiency, phosphoenolpyruvatecarboxykinase deficiency, and lactate dehydrogenase deficiency.

Nucleic acids and polypeptides that can be delivered to cardiac muscleinclude those that are beneficial in the treatment of damaged,degenerated or atrophied cardiac muscle and/or congenital cardiacdefects. For example, angiogenic factors useful for facilitatingvascularization in the treatment of heart disease include but are notlimited to vascular endothelial growth factor (VEGF), VEGF II, VEGF-B,VEGF-C, VEGF-D, VEGF-E, VEGF₁₂₁, VEGF₁₃₈, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉,VEGF₂₀₆, hypoxia inducible factor 1α (HIF 1α), endothelial NO synthase(eNOS), iNOS, VEFGR-1 (Flt1), VEGFR-2 (KDR/Flk1), VEGFR-3 (Flt4),angiogenin, epidermal growth factor (EGF), angiopoietin,platelet-derived growth factor, angiogenic factor, transforming growthfactor-α (TGF-α), transforming growth factor-β (TGF-β), vascularpermeability factor (VPF), tumor necrosis factor alpha (TNF-α),interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived endothelialgrowth factor (PD-EGF), granulocyte colony stimulating factor (G-CSF),hepatocyte growth factor (HGF), scatter factor (SF), pleitrophin,proliferin, follistatin, placental growth factor (PIGF), midkine,platelet-derived growth factor-BB (PDGF), fractalkine, ICAM-1,angiopoietin-1 and -2 (Ang1 and Ang2), Tie-2, neuropilin-1, ICAM-1,chemokines and cytokines that stimulate smooth muscle cell, monocyte, orleukocyte migration, anti-apoptotic peptides and proteins, fibroblastgrowth factors (FGF), FGF-1, FGF-1b, FGF-1c, FGF-2, FGF-2b, FGF-2c,FGF-3, FGF-3b, FGF-3c, FGF-4, FGF-5, FGF-7, FGF-9, acidic FGF, basicFGF, monocyte chemotactic protein-1, granulocyte macrophage-colonystimulating factor, insulin-like growth factor-1 (IGF-1), IGF-2, earlygrowth response factor-1 (EGR-1), ETS-1, human tissue kallikrein (HK),matrix metalloproteinase, chymase, urokinase-type plasminogen activatorand heparinase. (see, e.g., U.S. Patent Application No. 20060287259 andU.S. Patent Application No. 20070059288).

The most common congenital heart disease found in adults is bicuspidaortic valve, whereas atrial septal defect is responsible for 30-40% ofcongenital heart disease seen in adults. The most common congenitalcardiac defect observed in the pediatric population is ventricularseptal defect. Other congenital heart diseases include Eisenmenger'ssyndrome, patent ductus arteriosus, pulmonary stenosis, coarctation ofthe aorta, transposition of the great arteries, tricuspid atresia,univentricular heart, Ebstein's anomaly, and double-outlet rightventricle. A number of studies have identified putative genetic lociassociated with one or more of these congenital heart diseases. Forexample, the putative gene(s) for congenital heart disease associatedwith Down syndrome is 21q22.2-q22.3, between ETS2 and MX1. Similarly,most cases of DiGeorge syndrome result from a deletion of chromosome22811.2 (the DiGeorge syndrome chromosome region, or DGCR). Severalgenes are lost in this deletion including the putative transcriptionfactor TUPLE1. This deletion is associated with a variety of phenotypes,e.g., Shprintzen syndrome; conotruncal anomaly face (or Takao syndrome);and isolated outflow tract defects of the heart including Tetralogy ofFallot, truncus arteriosus, and interrupted aortic arch. All of theforegoing disorders can be treated according to the present invention.

Other significant diseases of the heart and vascular system are alsobelieved to have a genetic, typically polygenic, etiological component.These diseases include, for example, hypoplastic left heart syndrome,cardiac valvular dysplasia, Pfeiffer cardiocranial syndrome,oculofaciocardiodental syndrome, Kapur-Toriello syndrome, Sonodasyndrome, Ohdo Blepharophimosis syndrome, heart-hand syndrome,Pierre-Robin syndrome, Hirschsprung disease, Kousseff syndrome, Grangeocclusive arterial syndrome, Kearns-Sayre syndrome, Kartagener syndrome,Alagille syndrome, Ritscher-Schinzel syndrome, Ivemark syndrome,Young-Simpson syndrome, hemochromatosis, Holzgreve syndrome, Barthsyndrome, Smith-Lemli-Opitz syndrome, glycogen storage disease,Gaucher-like disease, Fabry disease, Lowry-Maclean syndrome, Rettsyndrome, Opitz syndrome, Marfan syndrome, Miller-Dieker lissencephalysyndrome, mucopolysaccharidosis, Bruada syndrome, humerospinaldysostosis, Phaver syndrome, McDonough syndrome, Marfanoid hypermobilitysyndrome, atransferrinemia, Cornelia de Lange syndrome, Leopardsyndrome, Diamond-Blackfan anemia, Steinfeld syndrome, progeria, andWilliams-Beuren syndrome. All of these disorders can be treatedaccording to the present invention.

Anti-apoptotic factors can be delivered to skeletal muscle, diaphragmmuscle and/or cardiac muscle to treat muscle wasting diseases, limbischemia, cardiac infarction, heart failure, coronary artery diseaseand/or type I or type II diabetes.

Nucleic acids that can be delivered to skeletal muscle include thosethat are beneficial in the treatment of damaged, degenerated and/oratrophied skeletal muscle. The genetic defects that cause musculardystrophy are known for many forms of the disease. These defective geneseither fail to produce a protein product, produce a protein product thatfails to function properly, or produce a dysfunctional protein productthat interferes with the proper function of the cell. The heterologousnucleic acid may encode a therapeutically functional protein or apolynucleotide that inhibits production or activity of a dysfunctionalprotein. Polypeptides that may be expressed from delivered nucleicacids, or inhibited by delivered nucleic acids (e.g., by deliveringRNAi, microRNA or antisense RNA), include without limitation dystrophin,a mini-dystrophin or a micro-dystrophin (Duchene's and Becker M D);dystrophin-associated glycoproteins β-sarcoglycan (limb-girdle MD 2E),δ-sarcoglycan (limb-girdle MD 2 2F), α-sarcoglycan (limb girdle MD 2D)and γ-sarcoglycan (limb-girdle MD 2C), utrophin, calpain (autosomalrecessive limb-girdle MD type 2A), caveolin-3 (autosomal-dominantlimb-girdle MD), laminin-alpha2 (merosin-deficient congenital MD),miniagrin (laminin-alpha2 deficient congenital MD), fukutin (Fukuyamatype congenital MD), emerin (Emery-Dreifuss MD), myotilin, lamin NC,calpain-3, dysferlin, and/or telethonin. Further, the heterologousnucleic acid can encode mir-1, mir-133, mir-206, mir-208 or an antisenseRNA, RNAi (e.g., siRNA or shRNA) or microRNA to induce exon skipping ina defective dystrophin gene.

In particular embodiments, the nucleic acid is delivered to tonguemuscle (e.g., to treat dystrophic tongue). Methods of delivering to thetongue can be by any method known in the art including direct injection,oral administration, topical administration to the tongue, intravenousadministration, intra-articular administration and the like.

The foregoing proteins can also be administered to diaphragm muscle totreat muscular dystrophy.

Alternatively, a gene transfer vector may be administered that encodesany other therapeutic polypeptide.

In particular embodiments, a virus vector according to the presentinvention is used to deliver a nucleic acid of interest as describedherein to skeletal muscle, diaphragm muscle and/or cardiac muscle, forexample, to treat a disorder associated with one or more of thesetissues such as muscular dystrophy, heart disease (including PAD andcongestive heart failure), and the like.

Gene transfer has substantial potential use in understanding andproviding therapy for disease states. There are a number of inheriteddiseases in which defective genes are known and have been cloned. Ingeneral, the above disease states fall into two classes: deficiencystates, usually of enzymes, which are generally inherited in a recessivemanner, and unbalanced states, which may involve regulatory orstructural proteins, and which are typically inherited in a dominantmanner. For deficiency state diseases, gene transfer can be used tobring a normal gene into affected tissues for replacement therapy, aswell as to create animal models for the disease using inhibitory RNAsuch as RNAi (e.g., siRNA or shRNA), microRNA or antisense RNA. Forunbalanced disease states, gene transfer can be used to create a diseasestate in a model system, which can then be used in efforts to counteractthe disease state. Thus, the virus vectors according to the presentinvention permit the treatment of genetic diseases. As used herein, adisease state is treated by partially or wholly remedying the deficiencyor imbalance that causes the disease or makes it more severe. The use ofsite-specific recombination of nucleic sequences to cause mutations orto correct defects is also possible.

The virus vectors according to the present invention may also beemployed to provide an antisense nucleic acid or inhibitory RNA (e.g.,microRNA or RNAi such as a siRNA or shRNA) to a cell in vitro or invivo. Expression of the inhibitory RNA in the target cell diminishesexpression of a particular protein(s) by the cell. Accordingly,inhibitory RNA may be administered to decrease expression of aparticular protein in a subject in need thereof. Inhibitory RNA may alsobe administered to cells in vitro to regulate cell physiology, e.g., tooptimize cell or tissue culture systems.

Further, the virus vectors according to the present invention findfurther use in diagnostic and screening methods, whereby a gene ofinterest is transiently or stably expressed in a cell culture system, oralternatively, a transgenic animal model. The invention can also bepracticed to deliver a nucleic acid for the purposes of proteinproduction, e.g., for laboratory, industrial or commercial purposes.

As a further aspect, the virus vectors of the present invention may beused to produce an immune response in a subject. According to thisembodiment, a virus vector comprising a nucleic acid encoding animmunogen may be administered to a subject, and an active immuneresponse (optionally, a protective immune response) is mounted by thesubject against the immunogen. Immunogens are as described hereinabove.

Alternatively, the virus vector may be administered to a cell ex vivoand the altered cell is administered to the subject. The heterologousnucleic acid is introduced into the cell, and the cell is administeredto the subject, where the heterologous nucleic acid encoding theimmunogen is optionally expressed and induces an immune response in thesubject against the immunogen. In particular embodiments, the cell is anantigen-presenting cell (e.g., a dendritic cell).

An “active immune response” or “active immunity” is characterized by“participation of host tissues and cells after an encounter with theimmunogen. It involves differentiation and proliferation ofimmunocompetent cells in lymphoreticular tissues, which lead tosynthesis of antibody or the development of cell-mediated reactivity, orboth.” Herbert B. Herscowitz, Immunophysiology: Cell Function andCellular Interactions in Antibody Formation, in IMMUNOLOGY: BASICPROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, anactive immune response is mounted by the host after exposure toimmunogens by infection or by vaccination. Active immunity can becontrasted with passive immunity, which is acquired through the“transfer of preformed substances (antibody, transfer factor, thymicgraft, interleukin-2) from an actively immunized host to a non-immunehost.” Id.

A “protective” immune response or “protective” immunity as used hereinindicates that the immune response confers some benefit to the subjectin that it prevents or reduces the incidence of disease. Alternatively,a protective immune response or protective immunity may be useful in thetreatment of disease, in particular cancer or tumors (e.g., by causingregression of a cancer or tumor and/or by preventing metastasis and/orby preventing growth of metastatic nodules). The protective effects maybe complete or partial, as long as the benefits of the treatmentoutweigh any disadvantages thereof.

The virus vectors of the present invention may also be administered forcancer immunotherapy by administration of a viral vector expressing acancer cell antigen (or an immunologically similar molecule) or anyother immunogen that produces an immune response against a cancer cell.To illustrate, an immune response may be produced against a cancer cellantigen in a subject by administering a viral vector comprising aheterologous nucleotide sequence encoding the cancer cell antigen, forexample to treat a patient with cancer. The virus vector may beadministered to a subject in vivo or by using ex vivo methods, asdescribed herein.

As used herein, the term “cancer” encompasses tumor-forming cancers.Likewise, the term “cancerous tissue” encompasses tumors. A “cancer cellantigen” encompasses tumor antigens.

The term “cancer” has its understood meaning in the art, for example, anuncontrolled growth of tissue that has the potential to spread todistant sites of the body (i.e., metastasize). Exemplary cancersinclude, but are not limited to, leukemia, lymphoma (e.g., Hodgkin andnon-Hodgkin lymphomas), colorectal cancer, renal cancer, liver cancer,breast cancer, lung cancer, prostate cancer, testicular cancer, ovariancancer, uterine cancer, cervical cancer, brain cancer (e.g.; gliomas andglioblastoma), bone cancer, sarcoma, melanoma, head and neck cancer,esophageal cancer, thyroid cancer, and the like. In embodiments of theinvention, the invention is practiced to treat and/or preventtumor-forming cancers.

The term “tumor” is also understood in the art, for example, as anabnormal mass of undifferentiated cells within a multicellular organism.Tumors can be malignant or benign. In representative embodiments, themethods disclosed herein are used to prevent and treat malignant tumors.

Cancer cell antigens have been described hereinabove. By the terms“treating cancer” or “treatment of cancer,” it is intended that theseverity of the cancer is reduced or the cancer is prevented or at leastpartially eliminated. For example, in particular contexts, these termsindicate that metastasis of the cancer is prevented or reduced or atleast partially eliminated. In further representative embodiments theseterms indicate that growth of metastatic nodules (e.g., after surgicalremoval of a primary tumor) is prevented or reduced or at leastpartially eliminated. By the terms “prevention of cancer” or “preventingcancer” it is intended that the methods at least partially eliminate orreduce the incidence or onset of cancer. Alternatively stated, the onsetor progression of cancer in the subject may be slowed, controlled,decreased in likelihood or probability, or delayed.

In particular embodiments, cells may be removed from a subject withcancer and contacted with a virus vector according to the presentinvention. The modified cell is then administered to the subject,whereby an immune response against the cancer cell antigen is elicited.This method is particularly advantageously employed withimmunocompromised subjects that cannot mount a sufficient immuneresponse in vivo (i.e., cannot produce enhancing antibodies insufficient quantities).

It is known in the art that immune responses may be enhanced byimmunomodulatory cytokines (e.g., α-interferon, β-interferon,γ-interferon, ω-interferon, τ-interferon, interleukin-1α,interleukin-1β, interleukin-2, interleukin-3, interleukin-4, interleukin5, interleukin-6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin 12, interleukin-13,interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand, tumornecrosis factor-α, tumor necrosis factor-β, monocyte chemoattractantprotein-1, granulocyte-macrophage colony stimulating factor, andlymphotoxin). Accordingly, immunomodulatory cytokines (e.g., CTLinductive cytokines) may be administered to a subject in conjunctionwith the virus vectors.

Cytokines may be administered by any method known in the art. Exogenouscytokines may be administered to the subject, or alternatively, anucleotide sequence encoding a cytokine may be delivered to the subjectusing a suitable vector, and the cytokine produced in vivo.

Recombinant virus vectors according to the present invention find use inboth veterinary and medical applications. Suitable subjects include bothavians and mammals. The term “avian” as used herein includes, but is notlimited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots,parakeets. The term “mammal” as used herein includes, but is not limitedto, humans, primates non-human primates (e.g., monkeys and baboons),cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g.,rats, mice, hamsters, and the like), etc. Human subjects includeneonates, infants, juveniles, and adults. Optionally, the subject is “inneed of the methods of the present invention, e.g., because the subjecthas or is believed at risk for a disorder including those describedherein or that would benefit from the delivery of a nucleic acidincluding those described herein. For example, in particularembodiments, the subject has (or has had) or is at risk for a musculardystrophy or heart disease (e.g., myocardial infarct, PAD, congestiveheart failure, etc.). As a further option, the subject can be alaboratory animal and/or an animal model of disease.

In particular embodiments, the present invention provides apharmaceutical composition comprising a virus vector of the invention ina pharmaceutically acceptable carrier and, optionally, other medicinalagents, pharmaceutical agents, stabilizing agents, buffers, carriers,adjuvants, diluents, etc. For injection, the carrier will typically be aliquid. For other methods of administration, the carrier may be eithersolid or liquid. For inhalation administration, the carrier will berespirable, and will preferably be in solid or liquid particulate form.

By “pharmaceutically acceptable” it is meant a material that is nottoxic or otherwise undesirable, i.e., the material may be administeredto a subject without causing any undesirable biological effects.

One aspect of the present invention is a method of transferring anucleotide sequence to a cell in vitro. The virus vector may beintroduced to the cells at the appropriate multiplicity of infectionaccording to standard transduction methods appropriate for theparticular target cells. Titers of the virus vector or capsid toadminister can vary, depending upon the target cell type and number, andthe particular virus vector or capsid, and can be determined by those ofskill in the art without undue experimentation. In particularembodiments, at least about 10³ infectious units, more preferably atleast about 10⁵ infectious units are introduced to the cell.

The cell(s) to be introduced the virus vector may be of any type,including but not limited to neural cells (including cells of theperipheral and central nervous systems, in particular, brain cells suchas neurons, oligodendricytes, glial cells, astrocytes), lung cells,cells of the eye (including retinal cells, retinal pigment epithelium,and corneal cells), epithelial cells (e.g., gut and respiratoryepithelial cells), skeletal muscle cells (including myoblasts, myotubesand myofibers), diaphragm muscle cells, dendritic cells, pancreaticcells (including islet cells), hepatic cells, a cell of thegastrointestinal tract (including smooth muscle cells, epithelialcells), heart cells (including cardiomyocytes), bone cells (e.g., bonemarrow stem cells), hematopoietic stem cells, spleen cells,keratinocytes, fibroblasts, endothelial cells, prostate cells, jointcells (including, e.g., cartilage, meniscus, synovium and bone marrow),germ cells, and the like. Alternatively, the cell may be any progenitorcell. As a further alternative, the cell can be a stem cell (e.g.,neural stem cell, liver stem cell). As still a further alternative, thecell may be a cancer or tumor cell (cancers and tumors are describedabove). Moreover, the cells can be from any species of origin, asindicated above.

The virus vectors may be introduced to cells in vitro for the purpose ofadministering the modified cell to a subject. In particular embodiments,the cells have been removed from a subject, the virus vector isintroduced therein, and the cells are then replaced back into thesubject. Methods of removing cells from subject for treatment ex vivo,followed by introduction back into the subject are known in the art(see, e.g., U.S. Pat. No. 5,399,346). Alternatively, the recombinantvirus vector is introduced into cells from another subject, intocultured cells, or into cells from any other suitable source, and thecells are administered to a subject in need thereof.

Suitable cells for ex vivo gene therapy are as described above. Dosagesof the cells to administer to a subject will vary upon the age,condition and species of the subject, the type of cell, the nucleic acidbeing expressed by the cell, the mode of administration, and the like.Typically, at least about 10² to about 10⁸ or about 10³ to about 10⁶cells will be administered per dose in a pharmaceutically acceptablecarrier. In particular embodiments, the cells transduced with the virusvector are administered to the subject in an effective amount incombination with a pharmaceutical carrier.

In some embodiments, cells that have been transduced with the virusvector may be administered to elicit an immunogenic response against thedelivered polypeptide (e.g., expressed as a transgene or in the capsid).Typically, a quantity of cells expressing an effective amount of thepolypeptide in combination with a pharmaceutically acceptable carrier isadministered. Optionally, the dosage is sufficient to produce aprotective immune response (as defined above). The degree of protectionconferred need not be complete or permanent, as long as the benefits ofadministering the immunogenic polypeptide outweigh any disadvantagesthereof.

A further aspect of the invention is a method of administering the virusvectors or capsids of the invention to subjects. In particularembodiments, the method comprises a method of delivering a nucleic acidof interest to an animal subject, the method comprising: administeringan effective amount of a virus vector according to the invention to ananimal subject. Administration of the virus vectors of the presentinvention to a human subject or an animal in need thereof can be by anymeans known in the art. Optionally, the virus vector is delivered in aneffective dose in a pharmaceutically acceptable carrier.

The virus vectors of the invention can further be administered to asubject to elicit an immunogenic response (e.g., as a vaccine).Typically, vaccines of the present invention comprise an effectiveamount of virus in combination with a pharmaceutically acceptablecarrier. Optionally, the dosage is sufficient to produce a protectiveimmune response (as defined above). The degree of protection conferredneed not be complete or permanent, as long as the benefits ofadministering the immunogenic polypeptide outweigh any disadvantagesthereof. Subjects and immunogens are as described above.

Dosages of the virus vectors to be administered to a subject will dependupon the mode of administration, the disease or condition to be treated,the individual subject's condition, the particular virus vector, and thenucleic acid to be delivered, and can be determined in a routine manner.Exemplary doses for achieving therapeutic effects are virus titers of atleast about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10³, 10¹⁴, 10¹⁵transducing units or more, preferably about 10⁷ or 10⁸-10¹², 10¹³ or10¹⁴ transducing units, yet more preferably about 10¹² transducingunits.

In particular embodiments, more than one administration (e.g., two,three, four or more administrations) may be employed to achieve thedesired level of gene expression over a period of various intervals,e.g., daily, weekly, monthly, yearly, etc.

Exemplary modes of administration include oral, rectal, transmucosal,topical, intranasal, inhalation (e.g., via an aerosol), buccal (e.g.,sublingual), vaginal, intrathecal, intraocular, transdermal, in utero(or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal,intramuscular [including administration to skeletal, diaphragm and/orcardiac muscle], intradermal, intrapleural, intracerebral, andintraarticular), topical (e.g., to both skin and mucosal surfaces,including airway surfaces, and transdermal administration),intro-lymphatic, and the like, as well as direct tissue or organinjection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragmmuscle or brain). Administration can also be to a tumor (e.g., in or anear a tumor or a lymph node). The most suitable route in any given casewill depend on the nature and severity of the condition being treatedand on the nature of the particular vector that is being used.

Administration to skeletal muscle according to the present inventionincludes but is not limited to administration to skeletal muscles in thelimbs (e.g., upper arm, lower arm, upper leg, and/or lower leg), back,neck, head (e.g., tongue), thorax, abdomen, pelvis/perineum, and/ordigits. Suitable skeletal muscle tissues include but are not limited toabductor digiti minimi (in the hand), abductor digiti minimi (in thefoot), abductor hallucis, abductor ossis metatarsi quinti, abductorpollicis brevis, abductor pollicis longus, adductor brevis, adductorhallucis, adductor longus, adductor magnus, adductor pollicis, anconeus,anterior scalene, articularis genus, biceps brachii, biceps femoris,brachialis, brachioradialis, buccinator, coracobrachialis, corrugatorsupercilii, deltoid, depressor anguli oris, depressor labii inferioris,digastric, dorsal interossei (in the hand), dorsal interossei (in thefoot), extensor carpi radialis brevis, extensor carpi radialis longus,extensor carpi ulnaris, extensor digiti minimi, extensor digitorum,extensor digitorum brevis, extensor digitorum longus, extensor hallucisbrevis, extensor hallucis longus, extensor indicis, extensor pollicisbrevis, extensor pollicis longus, flexor carpi radialis, flexor carpiulnaris, flexor digiti minimi brevis (in the hand), flexor digiti minimibrevis (in the foot), flexor digitorum brevis, flexor digitorum longus,flexor digitorum profundus, flexor digitorum superficialis, flexorhallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexorpollicis longus, frontalis, gastrocnemius, geniohyoid, gluteus maximus,gluteus medius, gluteus minimus, gracilis, iliocostalis cervicis,iliocostalis lumborum, iliocostalis thoracis, illiacus, inferiorgemellus, inferior oblique, inferior rectus, infraspinatus,interspinalis, intertransversi, lateral pterygoid, lateral rectus,latissimus dorsi, levator anguli oris, levator labii; superioris,levator labii superioris alaeque nasi, levator palpebrae superioris,levator scapulae, long rotators, longissimus capitis, longissimuscervicis, longissimus thoracis, longus capitis, longus colli, lumbricals(in the hand), lumbricals (in the foot), masseter, medial pterygoid,medial rectus, middle scalene, multifidus, mylohyoid, obliquus capitisinferior, obliquus capitis superior, obturator externus, obturatorinternus, occipitalis, omohyoid, opponens digiti minimi, opponenspollicis, orbicularis oculi, orbicularis oris, palmar interossei,palmaris brevis, palmaris longus, pectineus, pectoralis major,pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius,piriformis, plantar interossei, plantaris, platysma, popliteus,posterior scalene, pronator quadratus, pronator teres, psoas major,quadratus femoris, quadratus plantae, rectus capitis anterior, rectuscapitis lateralis, rectus capitis posterior major, rectus capitisposterior minor, rectus femoris, rhomboid major, rhomboid minor,risorius, sartorius, scalenus minimus, semimembranosus, semispinaliscapitis, semispinalis cervicis, semispinalis thoracis, semitendinosus,serratus anterior, short rotators, soleus, spinalis capitis, spinaliscervicis, spinalis thoracis, splenius capitis, splenius cervicis,sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius,subscapularis, superior gemellus, superior oblique, superior rectus,supinator, supraspinatus, temporalis, tensor fascia lata, teres major,teres minor, thoracis, thyrohyoid, tibialis anterior, tibialisposterior, trapezius, triceps brachii, vastus intermedius, vastuslateralis, vastus medialis, zygomaticus major, and zygomaticus minor andany other suitable skeletal muscle as known in the art. In particularembodiments, the virus vector comprises, consists of, or consistsessentially of the M41 capsid (FIG. 3A-3C) or the H50 capsid (FIG.3TT-3VV).

The virus vector can be delivered to skeletal muscle by any suitablemethod including without limitation intravenous administration,intra-arterial administration, intraperitoneal administration, isolatedlimb perfusion (of leg and/or arm; see, e.g. Arruda et al., (2005) Blood105: 3458-3464), and/or direct intramuscular injection.

Administration to cardiac muscle includes without limitationadministration to the left atrium, right atrium, left ventricle, rightventricle and/or septum. The virus vector can be delivered to cardiacmuscle by any method known in the art including, e.g., intravenousadministration, intra-arterial administration such as intra-aorticadministration, direct cardiac injection (e.g., into left atrium, rightatrium, left ventricle, right ventricle), and/or coronary arteryperfusion. In particular embodiments, the virus vector comprises,consists of, or consists essentially of the M41 capsid (FIG. 3A-3C) orthe H50 capsid (FIG. 3TT-3VV).

Administration to diaphragm muscle can be by any suitable methodincluding intravenous administration, infra-arterial administration,and/or intra-peritoneal administration. In particular embodiments, thevirus vector comprises, consists of, or consists essentially of the M41capsid (FIG. 3A-3C) or the H50 capsid (FIG. 3TT-3VV).

Delivery to any of these tissues can also be achieved by delivering adepot comprising the virus vector, which can be implanted into theskeletal, cardiac and/or diaphragm muscle tissue or the tissue can becontacted with a film or other matrix comprising the virus vector.Examples of such implantable matrices or substrates are described inU.S. Pat. No. 7,201,898).

In particular embodiments, a virus vector according to the presentinvention is administered to skeletal muscle, diaphragm muscle and/orcardiac muscle (e.g., to treat muscular dystrophy, heart disease [forexample, PAD or congestive heart failure]). Optionally, the virus vectorcomprises, consists of, or consists essentially of the M41 capsid (FIG.3A-3C) or the H50 capsid (FIG. 3TT-3VV).

The invention can be used to treat disorders of skeletal, cardiac and/ordiaphragm muscle. Alternatively, the invention can be practiced todeliver a nucleic acid to skeletal, cardiac and/or diaphragm muscle,which is used as a platform for production of a protein product (e.g.,an enzyme) or non-translated RNA (e.g., RNAi, microRNA, antisense RNA)that normally circulates in the blood or for systemic delivery to othertissues to treat a disorder (e.g., a metabolic disorder, such asdiabetes (e.g., insulin), hemophilia (e.g., Factor IX or Factor VIII),or a lysosomal storage disorder (such as Gaucher's disease[glucocerebrosidase], Pompe disease [lysosomal acid α-glucosidase] orFabry disease [α-galactosidase A]) or a glycogen storage disorder (suchas Pompe disease [lysosomal acid α glucosidase]). Other suitableproteins for treating metabolic disorders are described above.

In a representative embodiment, the invention provides a method oftreating muscular dystrophy in a subject in need thereof, the methodcomprising: administering an effective amount of a virus vector of theinvention to a mammalian subject, wherein the virus vector comprises aheterologous nucleic acid effective to treat muscular dystrophy. In anexemplary embodiment, the method comprises: administering an effectiveamount of a virus vector of the invention to a mammalian subject,wherein the virus vector comprises a heterologous nucleic acid encodingdystrophin, a mini-dystrophin, a micro-dystrophin, utrophin,mini-utrophin, laminin-α2, mini-agrin, Fukutin-related protein,follistatin, dominant negative myostatin, α-sarcoglycan, β-sarcoglycan,v-sarcoglycan, δ-sarcoglycan, IGF-1, myostatin pro-peptide, activin typeII soluble receptor, anti-inflammatory polypeptides such as the Ikappa Bdominant mutant, sarcospan, antibodies or antibody fragments againstmyostatin or myostatin propeptide, or inhibitory RNA (e.g., antisenseRNA, microRNA or RNAi) against myostatin, mir-1, mir-133, mir-206,mir-208 or an inhibitory RNA (e.g., microRNA, RNAi or antisense RNA) toinduce exon skipping in a defective dystrophin gene. In particularembodiments, the virus vector can be administered to skeletal, diaphragmand/or cardiac muscle as described elsewhere herein. Optionally, thevirus vector comprises, consists of, or consists essentially of the M41capsid (FIG. 3A-3C) or the H50 capsid (FIG. 3TT-3VV).

The invention further encompasses a method of treating a metabolicdisorder in a subject in need thereof. In representative embodiments,the method comprises: administering an effective amount of a virusvector of the invention to skeletal muscle of a subject, wherein thevirus vector comprises a heterologous nucleic acid encoding apolypeptide, wherein the metabolic disorder is a result of a deficiencyand/or defect in the polypeptide. Illustrative metabolic disorders andheterologous nucleic acids encoding polypeptides are described herein.Optionally, the virus vector comprises, consists of, or consistsessentially of the M41 capsid (FIG. 3A-3C) or the H50 capsid (FIG.3TT-3VV). As a further option, the heterologous nucleic acid can encodea secreted protein.

The invention can also be practiced to produce inhibitory RNA (e.g.,antisense RNA, microRNA or RNAi) for systemic delivery.

The invention also provides a method of treating congenital heartfailure in a subject in need thereof, the method comprisingadministering an effective amount of a virus vector of the invention toa mammalian subject, wherein the virus vector comprises a heterologousnucleic acid effective to treat congenital heart failure. Inrepresentative embodiments, the method comprises administering aneffective amount of a virus vector of the invention to a mammaliansubject, wherein the virus vector comprises a heterologous nucleic acidencoding a sarcoplasmic endoreticulum Ca²⁺-ATPase (SERCA2a), anangiogenic factor, phospholamban, PI3 kinase, calsarcan, a β-adrenergicreceptor kinase (βARK), βARKct, inhibitor 1 of protein phosphatase 1,Pim-1, PGC-1α, SOD-1, SOD-2, EC-SOD, Kallikrein, HIF, thymosin-β4,S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that effectsG-protein coupled receptor kinase type 2 knockdown such as a truncatedconstitutively active bARKct; phospholamban inhibitory ordominant-negative molecules such as phospholamban S16E, mir-1, mir-133,mir-206, mir-208. Optionally, the virus vector comprises, consists of,or consists essentially of the M41 capsid (FIG. 3A-3C) or the H50 capsid(FIG. 3TT-3VV).

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Alternatively,one may administer the virus vector in a local rather than systemicmanner, for example, in a depot or sustained-release formulation.Further, the virus vector can be delivered dried to a surgicallyimplantable matrix such as a bone graft substitute, a suture, a stent,and the like (e.g., as described in U.S. Pat. No. 7,201,898).

Pharmaceutical compositions suitable for oral administration can bepresented in discrete units, such as capsules, cachets, lozenges, ortablets, each containing a predetermined amount of the composition ofthis invention; as a powder or granules; as a solution or a suspensionin an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil emulsion. Oral delivery can be performed by complexing avirus vector of the present invention to a carrier capable ofwithstanding degradation by digestive enzymes in the gut of an animal.Examples of such carriers include plastic capsules or tablets, as knownin the art. Such formulations are prepared by any suitable method ofpharmacy, which includes the step of bringing into association thecomposition and a suitable carrier (which may contain one or moreaccessory ingredients as noted above). In general, the pharmaceuticalcomposition according to embodiments of the present invention areprepared by uniformly and intimately admixing the composition with aliquid or finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet can be prepared bycompressing or molding a powder or granules containing the composition,optionally with one or more accessory ingredients. Compressed tabletsare prepared by compressing, in a suitable machine, the composition in afree-flowing form, such as a powder or granules optionally mixed with abinder, lubricant, inert diluent, and/or surface active/dispersingagent(s). Molded tablets are made by molding, in a suitable machine, thepowdered compound moistened with an inert liquid binder.

Pharmaceutical compositions suitable for buccal (sub-lingual)administration include lozenges comprising the composition of thisinvention in a flavored base, usually sucrose and acacia or tragacanth;and pastilles comprising the composition in an inert base such asgelatin and glycerin or sucrose and acacia.

Pharmaceutical compositions suitable for parenteral administration cancomprise sterile aqueous and non-aqueous injection solutions of thecomposition of this invention, which preparations are optionallyisotonic with the blood of the intended recipient. These preparationscan contain anti-oxidants, buffers, bacteriostats and solutes, whichrender the composition isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions, solutions andemulsions can include suspending agents and thickening agents. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The compositions can be presented in unit\dose or multi-dose containers,for example, in sealed ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, saline or water-for-injectionimmediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets of the kind previously described.For example, an injectable, stable, sterile composition of thisinvention in a unit dosage form in a sealed container can be provided.The composition can be provided in the form of a lyophilizate, which canbe reconstituted with a suitable pharmaceutically acceptable carrier toform a liquid composition suitable for injection into a subject. Theunit dosage form can be from about 1 μg to about 10 grams of thecomposition of this invention. When the composition is substantiallywater-insoluble, a sufficient amount of emulsifying agent, which isphysiologically acceptable, can be included in sufficient quantity toemulsify the composition in an aqueous carrier. One such usefulemulsifying agent is phosphatidyl choline.

Pharmaceutical compositions suitable for rectal administration can bepresented as unit dose suppositories. These can be prepared by admixingthe composition with one or more conventional solid carriers, such asfor example, cocoa butter and then shaping the resulting mixture.

Pharmaceutical compositions of this invention suitable for topicalapplication to the skin can take the form of an ointment, cream, lotion,paste, gel, spray, aerosol, or oil. Carriers that can be used include,but are not limited to, petroleum jelly, lanoline, polyethylene glycols,alcohols, transdermal enhancers, and combinations of two or morethereof. In some embodiments, for example, topical delivery can beperformed by mixing a pharmaceutical composition of the presentinvention with a lipophilic reagent (e.g., DMSO) that is capable ofpassing into the skin.

Pharmaceutical compositions suitable for transdermal administration canbe in the form of discrete patches adapted to remain in intimate contactwith the epidermis of the subject for a prolonged period of time.Compositions suitable for transdermal administration can also bedelivered by iontophoresis (see, for example, Pharmaceutical Research3:318 (1986)) and typically take the form of an optionally bufferedaqueous solution of the composition of this invention. Suitableformulations can comprise citrate or bis\tris buffer (pH 6) orethanol/water and can contain from 0.1 to 0.2M active ingredient.

The virus vectors disclosed herein may be administered to the lungs of asubject by any suitable means, for example, by administering an aerosolsuspension of respirable particles comprised of the virus vectors, whichthe subject inhales. The respirable particles may be liquid or solid.Aerosols of liquid particles comprising the virus vectors may beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer or an ultrasonic nebulizer, as is known to those of skill inthe art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particlescomprising the virus vectors may likewise be produced with any solidparticulate medicament aerosol generator, by techniques known in thepharmaceutical art.

III. DIRECTED EVOLUTION AND IN VIVO PANNING FOR VIRUS VECTORS COMPRISINGSCRAMBLED AAV CAPSIDS

The present invention also encompasses novel methods for creating a poolof viruses comprising scrambled AAV capsids (e.g., AAV particlescomprising the scrambled AAV capsids) and then selecting in vivo forscrambled capsids/viruses having one or more desired characteristics.Non-limiting examples of such characteristics include tropism profile,the ability to evade neutralizing antibodies, and improved intracellulartrafficking.

In a representative embodiment, the invention provides a method ofidentifying a virus vector (e.g., an AAV vector) or AAV capsid having atropism profile of interest, the method comprising (a) providing acollection of virus vectors (e.g., AAV particles), wherein each AAVvector within the collection comprises: (i) an AAV capsid comprising,consisting of, or consisting essentially of capsid proteins generated byscrambling the capsid coding sequences of two or more different AAV,wherein the capsid amino acid sequences of the two or more different AAVdiffer by at least two amino acids; and (ii) a viral vector genome(e.g., an AAV vector genome) comprising: an AAV cap coding sequenceencoding the AAV capsid of (i); an AAV rep coding sequence; and at leastone terminal repeat (e.g., a 5′ and/or 3′ terminal repeat such as, forexample, and AAV 5′ and/or 3′ terminal repeat) that functions with theRep protein(s) encoded by the AAV rep coding sequence; wherein the AAVcapsid encapsidates the vector genome; (b) administering the collectionof virus vectors to a subject; (c) recovering a plurality of virusvectors as virions or as viral vector genomes each encoding an AAVcapsid from a target tissue, thereby identifying a virus vector or AAVcapsid having a tropism of interest.

The invention can also be practiced to identify in vivo a scrambled AAVcapsid or virus particle comprising the same having the ability to evadeneutralizing antibodies (e.g., neutralizing antibodies found in humanserum). For example, in vivo screening for resistance to neutralizingantibodies can be carried out by injecting human IgGs (e.g., IVIG; apool of human IgGs) into a subject (e.g., a non-human mammaliansubject). The IVIG naturally contains a mixture of antibodies againstall of the common AAVs seen by the human population. Alternatively,specific neutralizing antibody(ies) can be administered to the subject.The scrambled virus library is then injected into the subject andselection is carried out for viral genomes that enter the targettissue(s) of interest (e.g., heart, skeletal muscle, liver, etc). Thosegenomes that are isolated from the target tissue correspond to thosecapsids that are able to evade neutralization.

Thus, in representative embodiments, the invention provides a method ofidentifying a scrambled AAV capsid or virus vector (e.g., an AAV vector)comprising the same having the ability to evade neutralizing IgGs (e.g.,neutralizing human IgGs), the method comprising (a) administering a poolof IgGs (e.g., human IgGs) to a mammalian subject; (b) providing acollection of scrambled virus vectors (e.g., scrambled AAV vectors),wherein each virus vector within the collection comprises: (i) an AAVcapsid comprising a capsid protein generated by scrambling the capsidsequences of two or more different AAV, wherein the capsid amino acidsequences of the two or more different AAV differ by at least two aminoacids; and (ii) a viral vector genome (e.g., an AAV vector genome)comprising: a cap coding sequence encoding the AAV capsid of (i); an AAVrep coding sequence; and at least one terminal repeat (e.g., a 5′ and/or3′ terminal repeat such as for example, a 5′ and/or 3′ AAV terminalrepeat) that functions with the Rep protein(s) encoded by the AAV repcoding sequence; wherein the AAV capsid encapsidates the virus vectorgenome; (c) administering the collection of virus vectors to a subject;(d) recovering a plurality of virus vectors as virions or as viralvector genomes each encoding an AAV capsid from a target tissue, therebyidentifying a virus vector or AAV capsid having the ability to evadeneutralizing IgGs (e.g., neutralizing human IgGs). In particularembodiments, the pool of IgGs are an IVIG preparation.

By “evade” neutralizing antibodies, it is intended that there is atleast a partial reduction in neutralization as compared with a suitablecontrol (e.g., a naturally occurring AAV such as AAV2, AAV8 or AAV9),but the degree of evasion or “resistance” need not be complete as longas there is some reduction in neutralization as compared with thecontrol and as long as some of the vector is able to reach and transducethe target tissue.

In particular embodiments, the target tissue is skeletal muscle (asdescribed above), liver, cardiac muscle, diaphragm muscle, kidney,liver, pancreas, spleen, the gastrointestinal tract, lung, joint tissue,tongue, ovary, testis, germ cells, cancer cells, or a combination of theforegoing.

The sequences of any combination of two or more AAV capsids, whethernaturally-occurring or modified and whether now known or laterdiscovered can be “scrambled” or “shuffled” to create a collection ofAAV vectors comprising the scrambled capsids. In representativeembodiments, the collection of AAV vectors comprises capsids generatedby scrambling capsid sequences from two or more of the following: AAV1,AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, gooseAAV or snake AAV. As noted above, the collection of AAV capsids canfurther comprise non-naturally occurring AAV capsids, now known or laterdiscovered. Such modifications include substitutions includingsubstitutions of modified nucleic acids and/or amino acids, anddeletions including truncations and/or insertions (including peptidedisplay).

Further diversity among the scrambled capsids can be achieved by anymethod known in the art for introducing mutations into nucleic acidand/or amino acid sequences, e.g., using chemical mutagens, error-pronePCR, cassette mutagenesis, and the like.

In particular embodiments, the method is carried out as an iterativeprocess, e.g., steps (a) through (c) are carried out for two or moreiterative cycles. For example, one or more of the scrambled AAV capsidsequences identified in the first round of in vivo screening can be usedas a starting point to create a second (or subsequent) collection ofscrambled AAV capsids for in vivo selection.

Optionally, the in vivo selection methods can be combined with one ormore rounds of in vitro selection to further optimize the vectors. Forexample, in vivo selection can be carried out to identify scrambled AAVcapsids having a desired tropism profile and then in vitro selection canbe used to identify those scrambled AAV capsids having the ability toevade neutralization by antibodies (e.g., serum antibodies found in thehuman population).

The collection of AAV particles can be administered to the subject byany suitable method. In particular embodiments, the collection isadministered into the blood stream of the subject (e.g., intravenous orintra-articular).

Modes of administration and subjects are as described elsewhere herein.

The invention can be used to identify scrambled viruses or virus capsidshaving a desired tropism pattern or profile in vivo in a subject.Accordingly, in particular embodiments, the inventive methods compriserecovering AAV particles or viral genomes encoding the same from two ormore target tissues and identifying scrambled viruses or scrambled AAVcapsids having a desired tropism for the two or more target tissues. Forexample, in particular embodiments, a scrambled virus or scrambled AAVcapsid having efficient tropism for skeletal muscle, diaphragm muscleand/or cardiac muscle and inefficient tropism for liver, gonads and/orstem cells is identified.

The target cell or tissue (or one of the target cells/tissues) can alsobe a cancer cell or tumor tissue. For example, the scrambled virus canbe administered to an animal model of cancer and scrambled virusparticles or viral genomes encoding the same are isolated from cancercells or tumor. In representative embodiments, the animal model can beone that has an increased likelihood of forming cancers or tumors or canbe a xenograft model in which human tumor cells are grafted into theanimal.

Exemplary methods of DNA “scrambling, also called “shuffling,”“molecular breeding,” “fast forced evolution” and the like are known inthe art. See, e.g., U.S. Pat. No. 5,605,793; U.S. Pat. No. 6,165,793;U.S. Pat. No. 5,605,793; U.S. Pat. No. 6,117,679; Stemmer, (1994) Proc.Natl. Acad. Sci. 91:10747-10751; and Soong et al., (2000) NatureGenetics 25:436-439. Such methods have also been applied to directedevolution of viruses. See, e.g., U.S. Pat. No. 6,096,548 and U.S. Pat.No. 6,596,539. In a representative embodiment, a collection of AAVcapsid protein coding sequences (or portions thereof) are fragmented andrecombined in vitro by homologous and/or non-homologous recombination tocreate a collection of “scrambled” AAV capsid proteins. Scrambled virusis generated in which each scrambled capsid packages a nucleic acid(e.g., an AAV genome) comprising the corresponding capsid codingsequence (i.e., encoding the capsid protein of the virus). Thecollection of scrambled viruses is administered to a subject and in vivoselection is carried out for a characteristic of interest. For example,scrambled virus can be isolated from one or more target tissues toidentify those optimized capsid proteins having a desired tropismprofile. The method can be carried out in iterative cycles to furtheroptimize the capsid sequences.

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

EXAMPLES

The Examples below describe the modification of the AAV capsid gene bydirected evolution to generate an optimized vector for systemic genedelivery to heart and skeletal muscle. DNA shuffling was used to modifythe AAV capsid gene and an AAV library was constructed for screeningagainst a mouse model. AAV variants enriched in mouse heart and skeletalmuscle were isolated and characterized based on their tropism andneutralization properties. In general, this approach mimics the naturalevolution of a virus in an animal model.

Example 1 Materials & Methods

Generation of Chimeric AAV Library with Shuffled Capsid Genes

For construction of random chimeric AAV capsid gene libraries, AAVserotypes 1, 2, 3B, 4, 6, 7, 8, 9 were used as PCR templates. The capsidgenes were amplified by primers CAP-5′(5′-CCC-AAGCTTCGATCAACTACGCAGACAGGTACCAA-3′; SEQ ID NO:139) and CAP-3′(5′-ATAAGAAT-GCGGCCGC-AGAGACCAAAGTTCAACTGAAACGA-3′; SEQ ID NO:140) andmixed in equal ratio for DNA shuffling (Soong et al., (2000) Nat Genet.25: 436-9). In brief, 4 μg of the DNA templates were treated by 0.04 Uof DNase I at 15° C. briefly. DNA fragments in size of 300-1000 by werepurified by agarose gel electrophoresis, denatured, re-annealed andrepaired by pfu DNA polymerase to reassemble random capsid genes.Amplification was done by use of pfu DNA polymerase and CAP5′/CAP3′primers. The PCR program was 30 cycles of 94° C. 1 min, 60° C. 1 min and72° C. 4.5 min. The PCR products were then digested with Hind III andNot I and ligated into a Hind III and Not I digested plasmid backbonecontaining AAV2 Rep gene and inverted terminal repeats. The randominfectious plasmids library was obtained by transforming the aboveligated DNA into DH10B E. coli cells. Random clones were picked forrestriction enzyme analysis and replication and packaging viability in293 cells. The shuffled infectious AAV library was finally producedusing a self-packaging technique developed by Muller et al. (NatureBiotechnol. (2003) 21:1040-1046).

In Vivo Biopanning of Mutant AAV Capsid Library in Mice

A dose of 5×10¹¹ vector genomes of the chimeric AAV library wereinjected into adult C57BL/6J mice via tail vein. Three days later, micewere sacrificed and perfused with PBS to remove the blood from tissues.The hind limb skeletal muscles and liver were collected for total DNAisolation. The capsid genes enriched in the muscle were retrieved by PCRamplification using primers Cap 5′ and Cap 3′ and the iProof DNApolymerase (Bio-Rad). The PCR products were digested with Hind III andNot I and cloned similarly as described in previous section. The 43representative AAV capsid genotypes were identified from thereconstructed plasmid library by restriction analysis and mixed in equalratio for production of a secondary AAV library as described earlier.This AAV library was again injected i.v. in mice for secondary screeningand retrieval from muscle and liver. Random colonies were sequenced andcompared for their tissue distribution.

Sequence and Structure Analyses of Modified AAV Capsid Genes

Identification and alignment of the capsid genes from the mouse tissueswas done using Clustal X (Larkin et al. (2007) Clustal w and clustal xversion 2.0. Bioinformatics 23: 2947-8).

Tissue Tropism of AAV Vectors in Mice after Systemic Administration

The M41 capsid gene was used to package CMV-luciferase, CB-LacZ orCMV-LacZ reporter vectors for comparison with the AAV9 or AAV6 packagedones. 3×10¹¹ v.g. of reporter vectors containing CMV-luciferase orCB-LacZ genes were injected i.v. in 6 to 8-week-old C57BU6J mice forsystemic gene delivery and expression. The heart, skeletal muscle andmain internal organs of mice were collected 2-weeks later. Reporter geneexpression was monitored by luciferase assay (Luciferase Assay System;Promega) or β-gal assay (Galacto-Light Plus™ system; AppliedBiosystems). Cryosectioning and X-gal staining were used to visualizecell that expressed the LacZ in the heart and muscle. Total DNA wasextracted from mouse tissues for quantitative detection of vector genomecopies by Taqman probes (Applied Biosystems) with a single-copyendogenous gene (glucagon gene) as the diploid cell number reference.

Transduction of Primary Cardiomyocytes or Skeletal Muscle by AAV Vectors

Rat neonatal cardiomyocytes were isolated and cultured as previouslyreported. 24 hr after pre-plating, rAAV9-, rM41- or rAAV6-CMV-lacZvectors were inoculated onto the cardiomyocytes in infectionmultiplicity of 3000. Cells were fixed for X-gal staining or β-gal assayafter another 96 hr to detect transgene expression. For skeletal muscletransduction, 50 μL of virus dilutes containing 5×10˜9 v.g. of rAAVvectors were intramuscularly injected into gastrocnemius muscles ofadult 7-week-old C57BJ/6L mice. 14 days later muscle tissues werecollected for detection of transgene expression by X-gal staining orβ-gal assay. Vector genome distribution in cardiomyocytes or skeletalmuscles was quantitated by real-time PCR as described.

Gene Transfer and Functional Assays in the Hamster Models

Tropism of the new vectors in hamsters was first investigated in thenormal F1B hamsters. A dose of 10¹² vector genomes of M41-CMV-lacZ orAAV9-CMV-lacZ was administered into 2- to 3-month-old male F1B hamstersvia the jugular vein. Three weeks later heart, skeletal muscles and theinternal organs including the liver were collected for histologicalstaining. The synthetic muscle-specific promoter (SYN) C5-12 was used toachieve strong and muscle-specific δ-SG transgene expression in the TO-2hamsters (Zhu et al., (2005) Circulation 112: 2650-9). A dose of 10¹²vector genomes of the M41-SYN-δSG vectors were administered into7-week-old male TO-2 hamsters intravenously with untreated TO-2 or withnormal F1B hamsters as the control groups. Three days before and onemonth after vector administration, blood samples were collected from thehamsters by cardiac puncture. Sera were prepared for the creatine kinase(CK) activity assay (TECO Diagnostics). Four months after vectoradministration, the hamsters were subject to echocardiography analysisto assess their cardiac and whole-body muscle functions as reported (Zhuet al., (2005) Circulation 112: 2650-9). δ-SG expression was detected byimmunofluorescence staining or Western blotting (Zhu et al., (2005)Circulation 112: 2650-9). H&E, Masson's trichrome and Von Kossastainings of the heart tissues were used for histology, fibrosis andcalcification respectively (Zhu et al., (2005) Circulation 112: 2650-9).

Example 2 Direct In Vivo Panning of DNA-Shuffled AAV Library forMuscle-Targeting Capsids

We constructed a chimeric AAV library by DNA shuffling of the capsidgenes of AAV1, 2, 3, 4, 6, 7, 8 and 9, in order to select forcombinations of characteristics. The infectious AAV library withshuffled capsid genes was packaged by the method of Muller et al.(Nature Biotechnol. (2003) 21:1040-1046). DNA analysis by restrictiondigestions on randomly picked mutant AAV clones showed unique patternsand indicated that the vast majority were recombinants and viable inproducing AAV particles (FIG. 1).

Although it is known to use in vitro cell culture systems to screen fordesirable mutant AAVs, here we solely relied on a direct in vivoscreening method, because no cell culture system could simultaneouslymimic the in vivo conditions (e.g., the tight endothelial lining, thedifferentiated muscle cells and the liver, etc.). We used adult mice forin vivo biopanning of the AAV library. Following tail vein injection ofa dose of 5×10¹¹ v.g. (viral genomes), the AAV cap genes that wereenriched in the muscles were retrieved by PCR amplification. In thefirst round of in vivo screening, 43 distinct AAV clones wereidentified. They were mixed in equal ratio for secondary AAV libraryproduction and second-round in vivo screening. The clones enriched inskeletal muscle but scarce in the liver were further characterized.

A clone named M41 appeared 12 times in 79 randomly picked clones fromthe skeletal muscle pool but was absent in the liver pool (FIG. 2).Sequence alignment of its capsid amino acid sequence showed that it is arecombinant of four parental AAV serotypes, AAV1, 6, 7 and 8, withsegments from AAV1, 8, and 7 in the N-terminal half of the capsid andAAV6, 1 and 6 in the C-terminal half (FIG. 3A). The sequence of the M41capsid gene and amino acid sequence are depicted in FIGS. 3B and 3C,respectively. The phylogenetic map, capsid coding and amino acidsequences of other AAV mutants isolated from skeletal muscle and heartlibraries are depicted in FIGS. 3D-3EEE.

Example 3 M41 Vector Preferentially Transduces Myocardium after SystemicAdministration

We next investigated systemic gene delivery efficiency and tissuetropism of AAVM41. The luciferase reporter gene was packaged into viralcapsids of M41, AAV9, and AAV6 for a side-by-side comparison in vivo. At2 weeks post i.v. injection in young adult C57BJ/6L mice (6-8 wk),luciferase activities and vector DNA copy numbers in various tissueswere analyzed. Consistent with previous reports (Inagaki et al. (2006)Mol Ther 14: 45-53), the AAV9 vector efficiently transduced mouse heart,skeletal muscles, and particularly the liver, which had the highestluciferase activity (FIG. 4A) and vector DNA copy numbers (FIG. 4B).Similar to AAV9, the M41 vector also transduced the heart efficientlywith slightly lower luciferase activity and vector copy numbers (FIG.4B). In contrast, M41 showed dramatically reduced gene transfer in theliver, with the luciferase activity 81.1 fold lower and DNA copy number11.3 fold lower than AAV9. However, AAVM41 gene transfer in the skeletalmuscles, except in the tongue, was also significantly lower than AAV9(FIG. 4A). Interestingly, although the liver had higher DNA copy numbers(FIG. 4B), the heart showed the highest luciferase activity among alltissues examined in M41 injected mice (FIG. 4A), suggesting differentialintracellular trafficking and uncoating processes of AAVM41 in these twotissues.

Similar side-by-side comparison between AAVM41 and AAV6 at 2 weeks afteri.v. injection revealed higher gene transfer by AAVM41 in all skeletalmuscles, and dramatically higher gene transfer (>13 fold) in the heart(FIG. 4C), but more than 50% reduction in the liver. Interestingly,although AAV6 had significantly higher vector DNA copy numbers thanAAVM41 in some muscle tissues such as the tibialis anterior andquadriceps (Ta and Qd in FIG. 4D), the gene expression levels were muchlower. The inconsistency in vector genome quantity and transgeneexpression between these two viruses suggests a more complex differencein vector bioavailability in the muscle tissues, such as transcytosisthrough endothelial lining and preferential infection of muscle ratherthan non-muscle cells.

Since one aim of this study was to reduce liver infectivity, we comparedratios of heart vs. liver gene expression for the above three AAVs.While the heart vs. liver ratio of AAVM41 was greater than 10:1, thisratio was reversed to 1:3 in AAV9 and 1:6 in AAV6 (FIG. 4E).Consistently, the ratios of heart vs. liver vector DNA copy numbers alsoshowed a similar trend to the luciferase activities among the three AAVs(FIG. 4F). These data thus demonstrated improved tropism to the heartand much reduced tropism to the liver by AAVM41.

We next used a β-galactosidase (LacZ) reporter gene to directlyvisualize transgene expression in cardiomyocytes and myofibers. At 2weeks post vector i.v. injection in adult C57BJ/6L mice, approximatelyone half of the cardiomyocytes in the heart showed positive X-galstaining in AAV9- and AAVM41-treated mice (FIG. 5). Similar test wasalso performed in hamsters, a different and larger species. At 3 weekspost i.v. injection into adult F1B hamsters, nearly 100% of thecardiomyocytes showed positive X-gal staining in both AAV9 and AAVM41treated groups (FIG. 5). Quantitative enzyme assays showed nearlyidentical levels of LacZ expression in the hearts of AAV9- andAAVM41-treated mice as well as hamsters (data not shown). In theskeletal muscles of the above mice and hamsters, however, AAVM41 wasmuch less efficient than AAV9 (FIG. 5B). These results are consistentwith those of luciferase reporter gene transfer, suggesting preferentialtargeting of AAVM41 to the myocardium.

Example 4 Additional Iterations of In Vivo Screening

The coding sequences for the AAV capsids described in Example 2 wereused as templates for reshuffling. To illustrate, the AAV capsid clonesidentified by screening heart tissue were reshuffled to generate asecondary heart library. Similarly, a secondary skeletal muscle librarywas generated from the capsid mutants identified in skeletal muscle. Thesecondary heart library was subjected to three successive screenings toidentify those AAV capsid clones targeting heart (“HH” designation). Thesecondary skeletal muscle library was used for parallel screening forcapsid clones targeting heart (designated “MH”) and skeletal muscle(designated “MM”). FIGS. 6A to 6TTTT show the nucleic acid and aminoacid sequences of a representative number of AAV capsids identified fromthese three screens. In general, these particular clones exhibitedrelatively high frequency in heart or skeletal tissue and relatively lowfrequency in liver.

Example 5 Direct Infection of Cardiomyocytes and Skeletal Muscle by M41Vector

Since AAVM41 was initially isolated from the muscle but showed bestinfectivity to the heart after systemic delivery, we wished to examinethe direct infectivity of AAVM41 on primary cardiomyocyte culture or onskeletal muscles by intramuscular injection. The AAV-LacZ vectorspackaged by AAVM41, AAV6 and AAV9 were used to infect primarycardiomyocytes isolated from neonatal rats. Four days later, less than1% of the AAV9-infected cardiomyocytes expressed the LacZ gene, butapproximately 20% or 80% of the M41 or AAV6 cells expressed the LacZgene (FIG. 7A, top). Quantitative analysis showed that β-gal enzymeactivities of AAVM41- or AAV6-infected cardiomyocytes were 2.8- or6.2-fold of that of AAV9 (FIG. 7B). Similarly, the vector copy numbersin the cells were 2.3- and 10.3-fold of that of AAV9 (FIG. 7C). Thesedata indicated that AAVM41 infectivity for cardiomyocytes was higherthan AAV9 but lower than AAV6.

The above three AAV-LacZ vectors were then injected into mousegastrocnemius muscle for comparison of skeletal muscle infectivities.Two weeks later X-gal staining of the muscle cryosections revealed thestrongest expression by AAV6, intermediate expression by AAVM41 and weakexpression by AAV9 (FIG. 7A, lower panels). Quantitative p-galactivities of AAV6-injected muscles were 6.1-fold and 53.8-fold of thatof AAVM41 and AAV9 respectively (FIG. 7D). Together with the in vitrocardiomyocytes infection data, these results strongly suggest that themuch improved systemic muscle and heart gene delivery by AAVM41 overAAV6 is most likely due to improved capability of crossing the tightendothelial barrier and reaching muscle cells.

Example 6 Delivery of δ-Sarcoglycan Gene Via Mutant AAV Vectors

Since AAVM41 showed preferential gene transfer in the heart, we nextinvestigated the utility of this vector for gene therapy in a geneticmodel of cardiomyopathy, congestive heart failure and muscular dystrophymodel, the δ-sarcoglycan (δ-SG) deficient TO-2 hamster. The TO-2 hamsteris an animal model with a δ-sarcoglycan gene (δ-SG) deficiencymanifesting the limb girdle muscular dystrophy 2F and heart failure(Homburger, et al. (1966) NY Acad. Sci. 138:14-27). Systemic genetransfer of δ-SG can effectively ameliorate cardiac and skeletal musclepathology and profoundly improve function (Zhu, et al. (2005)Circulation 112:2650-2659).

Four months after i.v. injection of 1×10¹² v.g. of AAVM41-δ-SG vector,SG expression was detected predominantly in the heart byimmunofluorescent (IF) staining (FIG. 8A). Nearly 100% of thecardiomyocytes showed strong and uniform δ-SG expression. However, only10-30% of the skeletal muscle myofibers expressed δ-SG, as shown in theforelimb, tibialis anterior and tongue muscles. Western blottingconfirmed strongest expression of δ-SG in the heart (FIG. 8B). No δ-SGexpression was detected in the non-muscle tissues including the liver.The muscle-specific, heart-preferential transgene expression wasaccompanied by the lack of immune rejection or toxicity throughout theduration of the experiments.

We also evaluated the therapeutic efficacies of AAVM41-δ-SG treatment inthe TO-2 hamsters, which manifest both cardiomyopathy and musculardystrophy. First we measured serum levels of muscle creatine kinaseactivities and found no statistically significant difference between thetreated and untreated groups (data not shown), suggesting insufficienttherapeutic gene transfer in the skeletal muscles by AAVM41. This isconsistent with the IF staining results (FIG. 8A). We continued toexamine the therapeutic efficacy on cardiomyopathy. Upon necropsy, grossexamination of the untreated control TO-2 hamster hearts showed markedlydilation and prominent calcification plaques. In contrast, the hearts ofthe treated TO-2 hamsters exhibited normal gross morphology, similar tothose of wild-type control F1B hamsters (data not shown). Histologicalstaining further revealed large areas of cardiomyocyte degeneration(FIG. 8C, left panel), fibrosis (FIG. 8C, middle panel) or calcification(FIG. 8C, right panel) in the untreated TO-2 hamsters. However, thosepathological signs were dramatically reduced or completely diminished inthe AAVM41-treated hamster hearts. Echocardiography examination of thetreated TO-2 hamsters also showed great improvement on all majorparameters of cardiac functions, including left ventricle end-systolicdimension, percent fractional shortening, and left ventricle posteriorwall thickness, nearly identical to those of wild-type F1B hamsters, butsignificantly different from those of the untreated TO-2 hamsters(P<0.05 by Student's t-test) (data not shown). These data furtherdemonstrated the therapeutic efficacy by AAVM41 gene delivery inimproving cardiac functions of TO-2 hamsters.

Example 7 Resistance of M41 to Pre-Existing Neutralizing Antibodies inPooled Human IgGs

We investigated AAVM41 for its resistance to preexisting neutralizingantibodies. Commercially available human IVIG (pooled human IgGs forintravenous use) was used as the source of antibodies. AAVM41 wascompared with AAV2, the best characterized serotype, and AAV8, a newisolate with low prevalence of pre-existing antibodies in humanpopulation. The AAV2-, AAV8-, and AAVM41-LacZ vectors were pre-incubatedwith serial dilutions of IVIG, inoculated on Huh7 cell culture for 4days and then assayed for LacZ expression as an indicator of vectorresistance to neutralization. At 1:64 dilution of IVIG, AAV2 infectivitydecreased to 33%±4% of its control without IVIG. But AAV8 and AAVM41infectivities remained at 94%±4% and 83%±1% of their controls. Even atthe highest IVIG concentration (1:8 dilution), AAV8 and AAVM41 stillretained 33%±12% and 26%±3% of their infectivities, while AAV2 wasnearly completely neutralized under the same condition (data not shown).

Example 8 Sustained Transgene Expression from AAV Mutant Vectors

AAV vector is well-known for its sustained transgene expression intransduced tissues. To determine whether this characteristic is retainedfor the optimized AAV vectors, time course experiments are conducted,wherein 3×10¹¹ vector genomes of modified rAAV vector are injected intoC57BL/6J mice by tail veil route, with rAAV9 vector as a control. At oneweek, two weeks, two months and five months after injection, a group ofmice is sacrificed for each virus. Tissues including heart, liver,diaphragm and tibialis anterior are used for luciferase assay and genecopy number detection for evaluation of the persistence of transgeneexpression mediated by the optimized AAV vectors.

Example 9

In Vivo Screening for Resistance to Neutralizing Antibodies

For in vivo screening for resistance to neutralizing antibodies, amixture of human IgGs (IVIG; a pool of IgG from thousands of donors) isinjected into nude mice. The IVIG naturally contains a mixture ofantibodies against all of the common AAVs seen by the human population.After IVIG into nude mice, the mutant AAV library is then injected intothe mice and selection is carried out for viral genomes that entered thetarget tissue(s) of interest (heart, skeletal muscle, liver, etc). Thosegenomes that are isolated from the target tissue correspond to thosecapsids that are resistant to neutralization.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A nucleic acid encoding an AAV capsid, the nucleic acid comprising an AAV capsid coding sequence selected from the group consisting of: (a) the nucleotide sequence of FIG. 3E (M17) (SEQ ID NO:1); (b) the nucleotide sequence of FIG. 3H (M22) (SEQ ID NO:3); (c) the nucleotide sequence of FIG. 3K (M35) (SEQ ID NO:5); (d) the nucleotide sequence of FIG. 3B (M41) (SEQ ID NO:7); (e) the nucleotide sequence of FIG. 3N (M42) (SEQ ID NO:9); (f) the nucleotide sequence of FIG. 3Q (M62) (SEQ ID NO:11); (g) the nucleotide sequence of FIG. 3T (M67) (SEQ ID NO:13); (h) the nucleotide sequence of FIG. 3W (M125) (SEQ ID NO:15); (i) the nucleotide sequence of FIG. 3Z (M148) (SEQ ID NO:17); (j) the nucleotide sequence of FIG. 3CC (M151) (SEQ ID NO:19); (k) the nucleotide sequence of FIG. 3FF (H18) (SEQ ID NO:21); (l) the nucleotide sequence of FIG. 3II (H34) (SEQ ID NO:23); (m) the nucleotide sequence of FIG. 3LL (H39) (SEQ ID NO:25); (n) the nucleotide sequence of FIG. 3OO (H40) (SEQ ID NO:27); (O) the nucleotide sequence of FIG. 3RR (H43) (SEQ ID NO:29); (p) the nucleotide sequence of FIG. 3UU (H50) (SEQ ID NO:31); (q) the nucleotide sequence of FIG. 3XX (H53) (SEQ ID NO:33); (r) the nucleotide sequence of FIG. 3AAA (H66) (SEQ ID NO:35); (s) the nucleotide sequence of FIG. 3DDD (H109) (SEQ ID NO:37); (t) the nucleotide sequence of FIG. 6A (HH1) (SEQ ID NO:39); (u) the nucleotide sequence of FIG. 6C(HH15) (SEQ ID NO:41); (vv) the nucleotide sequence of FIG. 6E (HH19) (SEQ ID NO:43); (ww) the nucleotide sequence of FIG. 6G (HH27) (SEQ ID NO:45); (xx) the nucleotide sequence of FIG. 6I (HH35) (SEQ ID NO:47); (yy) the nucleotide sequence of FIG. 6K (HH41) (SEQ ID NO:49); (zz) the nucleotide sequence of FIG. 6M (HH45) (SEQ ID NO:51); (aaa) the nucleotide sequence of FIG. 6O (HH53) (SEQ ID NO:53); (bbb) the nucleotide sequence of FIG. 6Q (HH67) (SEQ ID NO:55); (ccc) the nucleotide sequence of FIG. 6S(HH68) (SEQ ID NO:57); (ddd) the nucleotide sequence of FIG. 6U (HH75) (SEQ ID NO:59); (eee) the nucleotide sequence of FIG. 6W (HH87) (SEQ ID NO:61); (fff) the nucleotide sequence of FIG. 6Y (HH64) (SEQ ID NO:63); (ggg) the nucleotide sequence of FIG. 6AA (MH4) (SEQ ID NO:65); (hhh) the nucleotide sequence of FIG. 6CC (MH18) (SEQ ID NO:67); (iii) the nucleotide sequence of FIG. 6EE (MH21) (SEQ ID NO:69); (jjj) the nucleotide sequence of FIG. 6GG (MH31) (SEQ ID NO:71); (kkk) the nucleotide sequence of FIG. 6II (MH39) (SEQ ID NO:73); (lll) the nucleotide sequence of FIG. 6KK (MHY43) (SEQ ID NO:75); (mmm) the nucleotide sequence of FIG. 6MM (MH47) (SEQ ID NO:77); (nnn) the nucleotide sequence of FIG. 6OO (MH58) (SEQ ID NO:79); (ooo) the nucleotide sequence of FIG. 6QQ (MH63) (SEQ ID NO:81); (ppp) the nucleotide sequence of FIG. 6SS (MH71) (SEQ ID NO:83); (qqq) the nucleotide sequence of FIG. 6UU (MH74) (SEQ ID NO:85); (rrr) the nucleotide sequence of FIG. 6WW (MH78) (SEQ ID NO:87); (sss) the nucleotide sequence of FIG. 6YY (MH82) (SEQ ID NO:89); (ttt) the nucleotide sequence of FIG. 6AAA (MH90) (SEQ ID NO:91); (uuu) the nucleotide sequence of FIG. 6CCC (MH94) (SEQ ID NO:93); (vvv) the nucleotide sequence of FIG. 6EEE (MH95) (SEQ ID NO:95); (www) the nucleotide sequence of FIG. 6GGG (MH107) (SEQ ID NO:97); (xxx) the nucleotide sequence of FIG. 6III (MH113) (SEQ ID NO:99); (yyy) the nucleotide sequence of FIG. 6KKK (MM4) (SEQ ID NO:101); (zzz) the nucleotide sequence of FIG. 6MMM (MM7) (SEQ ID NO:103); (aaaa) the nucleotide sequence of FIG. 6OOO (MM19) (SEQ ID NO:105); (bbbb) the nucleotide sequence of FIG. 6QQQ (MM35) (SEQ ID NO:107); (cccc) the nucleotide sequence of FIG. 6SSS (MM44) (SEQ ID NO:109); (dddd) the nucleotide sequence of FIG. 6OOO (MM55) (SEQ ID NO:111); (eeee) the nucleotide sequence of FIG. 6WWW (MM65) (SEQ ID NO:113); the nucleotide sequence of FIG. 6YYY (MM68) (SEQ ID NO:115); (gggg) the nucleotide sequence of 6AAAA (MM84) (SEQ ID NO:117); (hhhh) the nucleotide sequence of FIG. 6CCCC (MM107) (SEQ ID NO:119); (iiii) the nucleotide sequence of FIG. 6EEEE (MM112) (SEQ ID NO:121); (jjjj) the nucleotide sequence of FIG. 6GGGG (MM115) (SEQ ID NO:123); (kkkk) the nucleotide sequence of FIG. 6IIII (MM120) (SEQ ID NO:125); (llll) the nucleotide sequence of FIG. 6KKKK (MM123) (SEQ ID NO:127); (mmmm) the nucleotide sequence of FIG. 6MMMM (MM136) (SEQ ID NO:129); (nnnn) the nucleotide sequence of FIG. 6OOOO (MM138) (SEQ ID NO:131); (oooo) the nucleotide sequence of FIG. 6QQQQ (MM141) (SEQ ID NO:133); (pppp) the nucleotide sequence of FIG. 6SSSS (MM144) (SEQ ID NO:135), (qqqq) or the nucleotide sequence of FIG. 6UUUU (MM153) (SEQ ID NO:137); and (rrrr) a nucleotide sequence that encodes an AAV capsid encoded by the nucleotide sequence of any of (a) to (qqqq) but that differs from the nucleotide sequences of (a) to (qqqq) due to the degeneracy of the genetic code.
 2. The nucleic acid of claim 1, wherein the nucleic acid is a plasmid, phage, viral vector, bacterial artificial chromosome (BAC), or yeast artificial chromosome (YAC).
 3. The nucleic acid of claim 2, wherein the nucleic acid is an AAV vector comprising the coding sequence.
 4. The nucleic acid of claim 3, wherein the nucleic acid further comprises an AAV Rep coding sequence.
 5. A cell in vitro comprising the nucleic acid of claim 1 stably incorporated into the genome.
 6. A virus particle comprising the nucleic acid of claim
 1. 7. The virus particle of claim 6, wherein the virus particle is an AAV particle, an adenovirus particle, a herpesvirus particle, or a baculovirus particle.
 8. An AAV capsid encoded by the nucleic acid of claim
 1. 9. The AAV capsid of claim 8 covalently linked, bound to, or encapsidating a compound selected from the group consisting of a DNA molecule, an RNA molecule, a polypeptide, a carbohydrate, a lipid, and a small organic molecule.
 10. An AAV particle comprising: an AAV vector genome; and the AAV capsid of claim 8, wherein the AAV capsid encapsidates the AAV vector genome.
 11. The AAV particle of claim 10, wherein the AAV vector genome comprises a heterologous nucleic acid.
 12. The AAV particle of claim 11, wherein the heterologous nucleic acid encodes an antisense RNA, microRNA or RNAi.
 13. The AAV particle of claim 11, wherein the heterologous nucleic acid encodes a polypeptide.
 14. The AAV particle of claim 13, wherein the heterologous nucleic acid encodes an immunogen.
 15. The AAV particle of claim 13, wherein the heterologous nucleic acid encodes a therapeutic polypeptide.
 16. The AAV particle of claim 1, wherein the heterologous nucleic acid encodes dystrophin, a mini-dystrophin, a micro-dystrophin, a laminin-α2, a mini-agrin, an α-sarcoglycan, a β-sarcoglycan, a γ-sarcoglycan, a δ-sarcoglycan, utrophin, Fukutin-related protein, myostatin pro-peptide, follistatin, dominant negative myostatin, IGF-1, a sarcoplasmic endoreticulum Ca²⁺-ATPase (SERCA2a), a β-adrenergic receptor kinase inhibitor (βARKct), phospholamban, PI3 kinase, Pim-1, PGC-1a, SOD-1, SOD-2, EC-SOD, Kallikrein, HIF, thymosin-β4, mir-1, mir-133, mir-206, mir-208, inhibitor 1 of protein phosphatase 1, an anti-apoptotic factor, an angiogenic factor, insulin, Factor IX, Factor VIII, glucocerebrosidase, a-galactosidase A and/or lysosomal acid a glucosidase.
 17. The AAV particle of claim 11, wherein the heterologous nucleic acid encodes a reporter protein.
 18. A method of producing a recombinant AAV particle comprising an AAV capsid, the method comprising: providing a cell in vitro with a nucleic acid according to claim 1, an AAV rep coding sequence, an AAV vector genome comprising a heterologous nucleic acid, and helper functions for generating a productive AAV infection; and allowing assembly of the recombinant AAV particle comprising the AAV capsid and encapsidating the AAV vector genome.
 19. An AAV particle produced by the method of claim
 18. 20. A pharmaceutical formulation comprising the nucleic acid of claim 1 in a pharmaceutically acceptable carrier.
 21. A method of delivering a nucleic acid of interest to a cell, the method comprising administering the AAV particle of claim 10 to the cell.
 22. The method of claim 21, wherein the cell is a skeletal muscle cell, a cardiomyocyte, a diaphragm muscle cell, a pancreas cell, a spleen cell, a gastrointestinal cell, a lung cell, a joint cell, or a kidney cell.
 23. A method of delivering a nucleic acid of interest to a mammalian subject, the method comprising: administering an effective amount of the MV particle of claim 10 to a mammalian subject.
 24. The method of claim 23, wherein the mammalian subject is a human subject.
 25. The method of claim 23, wherein the AAV particle is delivered to skeletal muscle by intravenous administration, intra-arterial administration, intraperitoneal administration, isolated limb perfusion, or direct skeletal muscle injection.
 26. The method of claim 23, wherein the AAV particle is delivered to diaphragm muscle by intravenous administration, intra-arterial administration, and/or intra-peritoneal administration.
 27. The method of claim 23, wherein the AAV particle is delivered to cardiac muscle by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection, and/or coronary artery perfusion.
 28. A method of treating muscular dystrophy in a subject in need thereof, the method comprising: administering an effective amount of the AAV particle of claim 10 to the mammalian subject, wherein the AAV particle comprises a heterologous nucleic acid encoding dystrophin, a mini-dystrophin, a micro-dystrophin, a laminin-α2, a mini-agrin, an α-sarcoglycan, a β-sarcoglycan, a γ-sarcoglycan, a δ-sarcoglycan, utrophin, Fukutin-related protein, myostatin pro-peptide, follistatin, dominant negative myostatin, IGF-1, myostatin pro-peptide, mir-1, mir-133, mir-206, mir-208, an angiogenic factor, an anti-apoptotic factor, or any combination thereof.
 29. The method of claim 28, wherein the administration is by intravenous administration, intra-arterial administration including intra-aortic administration, intraperitoneal administration, isolated limb perfusion, direct skeletal muscle injection, intra-peritoneal administration, direct cardiac injection, or coronary artery perfusion.
 30. A method of treating congenital heart failure in a subject in need thereof, the method comprising: administering an effective amount of the AAV particle of claim 10 to the mammalian subject, wherein the AAV particle comprises a heterologous nucleic acid encoding a sarcoplasmic endoreticulum Ca²⁺-ATPase (SERCA2a), a β-adrenergicreceptor kinase inhibitor (βARKct), phospholamban, PI3 kinase, Pim-1, PGC-1α, SOD-1, SOD-2, EC-SOD, Kallikrein, HIF, thymosin-β4, mir-1, mir-133, mir-206, mir-208, inhibitor 1 of protein phosphatase 1, an anti-apoptotic factor, an angiogenic factor, or any combination thereof.
 31. The method of identifying an AAV vector or AAV capsid having a tropism profile of interest, the method comprising: (a) providing a collection of AAV vectors, wherein each AAV vector within the collection comprises: (i) an AAV capsid comprising capsid proteins generated by shuffling the capsid coding sequences of two or more different AAV, wherein the capsid amino acid sequences of the two or more different AAV differ by at least two amino acids; and (ii) an AAV vector genome comprising: a cap coding sequence encoding the AAV capsid of (i); an AAV rep coding sequence; and at least one terminal repeat that functions with the Rep protein(s) encoded by the AAV rep coding sequence; wherein the AAV capsid encapsidates the AAV vector genome; (b) administering the collection of AAV vectors to a mammalian subject; and (c) recovering a plurality of AAV vectors as virions or as viral genomes each encoding an AAV capsid from a target tissue, thereby identifying an AAV vector or AAV capsid having a tropism of interest.
 32. The method of claim 31, wherein the target tissue is skeletal muscle, liver, cardiac muscle, diaphragm, kidney, liver, pancreas, spleen, gastrointestinal tract, lung, joint tissue, tongue, ovary, testis, a germ cell, a cancer cell, or a combination of the foregoing.
 33. The method of claim 31, wherein the collection of AAV vectors comprises capsids generated by shuffling capsids from two or more of the following: AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goose AAV, or snake AAV.
 34. The method of claim 31, wherein the collection of AAV vectors is administered into the blood stream of the mammalian subject.
 35. The method of claim 31, wherein the method comprises recovering AAV vectors from two or more target tissues and identifying AAV vectors having a desired tropism for the two or more target tissues.
 36. The method of claim 35, wherein an AAV vector that efficiently transduces skeletal muscle and does not efficiently transduce liver is identified.
 37. The method of claim 35, wherein an AAV vector that efficiently transduces cardiac muscle and does not efficiently transduce liver is identified.
 38. The method of claim 35, wherein an AAV vector that efficiently transduces diaphragm muscle and does not efficiently transduce liver is identified.
 39. The method of claim 36, wherein the collection of AAV vectors is administered into the bloodstream of the mammalian subject.
 40. The method of claim 31, wherein the target tissue is a cancer tissue and the method is carried out to identify an AAV vector that has a desired tropism for the cancer tissue.
 41. The method of claim 31, wherein steps (a) through (c) are carried out for two or more iterative cycles.
 42. A pharmaceutical formulation comprising the virus particle of claim 6 in a pharmaceutically acceptable carrier.
 43. A pharmaceutical formulation comprising the AAV capsid of claim 9 in a pharmaceutically acceptable carrier.
 44. A pharmaceutical formulation comprising the AAV particle of claim 10 in a pharmaceutically acceptable carrier.
 45. A method of delivering a nucleic acid of interest to a mammalian subject, the method comprising: administering an effective amount of the pharmaceutical formulation of claim 20 to a mammalian subject.
 46. A method of treating muscular dystrophy in a subject in need thereof, the method comprising: administering an effective amount of the pharmaceutical formulation of claim 20 to the mammalian subject, wherein the AAV particle comprises a heterologous nucleic acid encoding dystrophin, a mini-dystrophin, a micro-dystrophin, a laminin-α2, a mini-agrin, an α-sarcoglycan, a β-sarcoglycan, a γ-sarcoglycan, a δ-sarcoglycan, utrophin, Fukutin-related protein, myostatin pro-peptide, follistatin, dominant negative myostatin, IGF-1, myostatin pro-peptide, mir-1, mir-133, mir-206, mir-208, an angiogenic factor, an anti-apoptotic factor, or any combination thereof.
 47. A method of treating congenital heart failure in a subject in need thereof, the method comprising: administering an effective amount of the pharmaceutical formulation of claim 20 to the mammalian subject, wherein the AAV particle comprises a heterologous nucleic acid encoding a sarcoplasmic endoreticulum Ca²⁺-ATPase (SERCA2a), a β-adrenergic receptor kinase inhibitor (βARKct), phospholamban, PI3 kinase, Pim-1, PGC-1α, SOD-1, SOD-2, EC-SOD, Kallikrein, HIF, thymosin-β4, mir-1, mir-133, mir-206, mir-208, inhibitor 1 of protein phosphatase 1, an anti-apoptotic factor, an angiogenic factor, or any combination thereof. 